Multiplanar Lateral Flow Assay with Sample Compressor

ABSTRACT

A sample compressor applies pressure to a sample collector and a sample application zone of a test strip to transfer a sample from the sample collector and a binding partner of an analyte to the sample application zone in a lateral flow device. At least one of the binding partners of the analyte is not located on the test strip prior to use of the lateral flow device. The test strip may be a universal test strip with no molecule that specifically binds the analyte is located on the test strip. The sample compressor may be a universal sample compressor also with no molecule that specifically binds the analyte on the sample compressor. The lateral flow device may also include one or more enhancement elements, where the enhancement elements bind to the analyte sandwich to increase a detection signal in the test zone.

REFERENCE TO RELATED APPLICATIONS

This application claims one or more inventions which were disclosed inProvisional Application No. 61/266,641, filed Dec. 4, 2009, entitled“LATERAL FLOW NUCLEIC ACID DETECTOR”, Provisional Application No.61/331,966, filed May 6, 2010, entitled “MULTIPLANAR LATERAL FLOW ASSAYWITH SAMPLE COMPRESSOR”, Provisional Application No. 61/352,093, filedJun. 7, 2010, entitled “LATERAL FLOW ASSAYS”, and ProvisionalApplication No. 61/392,981, filed Oct. 14, 2010, entitled “MULTIPLANARLATERAL FLOW ASSAY WITH SAMPLE COMPRESSOR”. The benefit under 35 USC§119(e) of the United States provisional applications is hereby claimed,and the aforementioned applications are hereby incorporated herein byreference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention pertains to the field of point of care tests. Moreparticularly, the invention pertains to lateral flow assays.

2. Description of Related Art

Lateral flow assays are a subset of assays combining various reagentsand process steps in one assay strip, thus providing a sensitive andrapid means for the detection of target molecules. Antibody-basedlateral flow immunoassays are available for a wide range of targetanalytes and can be designed for sandwich or competitive testprinciples. Generally, high molecular weight analytes with severalepitopes are analyzed in a sandwich format whereas small moleculesrepresenting only one epitope are detected by means of a competitiveassay. The first tests were made for human chorionic gonadotropin (hCG).Today there are commercially available tests for monitoring ovulation,detecting infectious disease organisms, analyzing drugs of abuse, andmeasuring other analytes important to human physiology. Products havealso been introduced for veterinary testing, environmental testing, andproduct monitoring.

In the prior art, the mobile labeled receptor (also known as the traceror the test conjugate herein) in these assays is either dried on thetest strip, contained in an external eluting solution (such that it canbe pre-mixed with the sample prior to application on the test strip), orpart of the elution media.

European patent publication EP0582231, published Feb. 9, 1994, entitled“SOLID PHASE ASSAY”, discloses an assay with a porous solid support witha first portion that contacts a sample that may include an analyte ofinterest. The sample flows through the solid support, and the analyte,if present, combines with a tracer, which is reversibly bound on thesolid support. The sample and the tracer initially travel in a directionperpendicular to the first portion (e.g. vertically) via capillary flow.The tracer and analyte then continue to travel by capillary flow throughthe material to a second portion that includes an immobilized binder,which binds to the analyte in a sandwich immunoassay format. Travel tothe second portion occurs in a direction perpendicular to the directionin which the tracer and sample initially travel (e.g. laterally). Alltravel of the sample and tracer occur due to capillary flow through thedevice. Although travel occurs vertically and laterally, there is asingle flow path. The sample, the tracer, and the immobilized binder areall in the same flow path.

U.S. Patent Publication No. 2007/0224701, published Sep. 27, 2007,entitled “COMBINATION VERTICAL AND LATERAL FLOW IMMUNOASSAY DEVICE”,discloses immunoassay devices, kits, and methods for determining thepresence or absence of an analyte in a liquid sample using a combinationof vertical flow and lateral flow. The device includes a tracer pad witha labeled receptor that is vertically juxtaposed with a binder supportmedium. The device disclosed in this publication is multi-sectioned,but, similar to EP0582231, only has a single flow path. The sample, thelabeled receptor, and the binder support medium are all in the same flowpath.

SUMMARY OF THE INVENTION

A sample compressor applies pressure to a sample collector at the sampleapplication zone of a test strip to transfer a sample on the samplecollector and a binding partner of an analyte to the sample applicationzone in a lateral flow device. At least one of the binding partners ofthe analyte is not located on the test strip or in the eluting solutionprior to use of the lateral flow device. The test strip may be auniversal test strip with no molecule that specifically binds theanalyte on the test strip. The sample compressor may be a universalsample compressor with no molecule that specifically binds the analyteon the sample compressor. The lateral flow device may also include anenhancement element, where the enhancement element binds to the analytesandwich to increase a detection signal in the test zone.

In one embodiment of the present invention, the lateral flow device fordetecting an analyte includes a sample compressor, a sample collectorwith a sample collection portion, a test strip with a sample applicationzone and a test zone, a conjugate including a first binding partner forthe analyte and a label, and a second binding partner for the analyte.Either the conjugate or the second binding partner or both the conjugateand the second binding partner are not located on the test strip priorto use of the lateral flow device. The sample compressor, the samplecollector, and the test strip form a vertical stack to apply the sampleto the test strip by compression. The sample compressor preferably has apad/fleece with the conjugate and/or the second binding partner beinglocated on the pad prior to use of the lateral flow device. In someembodiments, the lateral flow device includes a first control bindingpartner located on the sample compressor pad and a second controlbinding partner immobilized in a control zone of the test strip, wherethe first control binding partner is a binding partner for the secondcontrol binding partner. The lateral flow device is preferably formedsuch that a positive result is only achieved by isolation of the analytein the test zone by binding of the analyte to the first binding partnerand the second binding partner. The test zone preferably includes nomolecule which specifically binds the analyte. Preferably, the secondbinding partner includes a tag and the test zone includes an immobilizedbinding partner for the tag.

In another embodiment of the present invention, the universal test stripincludes a test zone but no molecule which specifically binds ananalyte. The test strip preferably also includes a control zone with acontrol binding partner immobilized in the control zone. The test strippreferably also includes a tag immobilized in the test zone, where thetag is biotin, avidin, neutravidin, streptavidin, a lectin, or aglycosyl moiety.

In yet another embodiment of the present invention, the samplecompressor for use in a lateral flow device includes a pad and at leastone binding partner of an analyte. The sample compressor preferably alsoincludes a mobile control binding partner on the pad.

In some embodiments, the sample compressor is a universal samplecompressor with no molecule which specifically binds an analyte. Theuniversal sample compressor preferably includes a pad and a mobilecontrol binding partner on the pad.

In another embodiment of the present invention, the lateral flow devicefor detecting an analyte includes a test strip with a sample applicationzone and a test zone, a conjugate with a first binding partner for theanalyte and a label, a second binding partner for the analyte, and anenhancement element. The analyte, the conjugate, and the second bindingpartner form a sandwich which is immobilized in the test zone when theanalyte is present, and the enhancement element binds to the sandwich toincrease a detection signal in the test zone. In some embodiments, theconjugate includes colloidal gold and the enhancement element includesat least one silver salt. In other embodiments, the enhancement elementincludes an antigen and the conjugate includes a specific bindingpartner for the antigen. The enhancement element preferably includes alabel. In a preferred embodiment, the test zone does not include amolecule which specifically binds the analyte. Preferably, the secondbinding partner includes a tag and the test zone includes an immobilizedbinding partner of the tag.

In yet another embodiment of the present invention, the method ofapplying a sample to a test strip of a lateral flow device includesplacing a sample collector with a sample collection portion with thesample in a vertical stack between a sample compressor and a sampleapplication zone of the test strip and applying a pressure to the samplecollection portion using the sample compressor to transfer at least aportion of the sample to the sample application zone. The methodpreferably includes placing a pad with a binding partner for an analyteon the vertical stack, and applying the pressure to transfer at least aportion of the binding partner to the sample application zone. Transferof the sample to the sample application zone preferably does not occurby flow.

In yet another embodiment of the present invention, the method ofapplying a sample to a test strip of a lateral flow device includesfirst placing at least one external binding partner on the sampleapplication zone of the test strip. The external binding partner may belocated on an external pad. In embodiments where there are two analytebinding partners that bind the analyte prior to reaching the test zone,either one or both of the analyte binding partners may be added. Asample collector that includes the sample is placed in a vertical stackbetween the external binding partner and a sample compressor. The samplecompressor applies pressure to the sample collector to transfer theexternal binding partner and at least a portion of the sample to thesample application zone. Alternatively, the external binding partnercould be added and compressed by the sample compressor, then removed,before the sample collector is stacked above the sample applicationzone, where the sample is compressed onto the test strip. In anotheralternative embodiment, at least one external binding partner is placedin the vertical stack between the sample compressor and samplecollector. Alternatively, the sample collector is added and compressed,then removed, and then the external binding partner is added andcompressed onto the test strip. In other embodiments, the samplecollector is in a vertical stack between a first external bindingpartner and a second external binding partner, and the sample compressorapplies pressure to the vertical stack. In these embodiments, neitherthe strip nor the sample compressor has a specific analyte bindingpartner.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a test strip and a sample collector in a lateral flowdevice.

FIG. 2A shows a sample compressor in an embodiment of the presentinvention.

FIG. 2B shows another sample compressor in an embodiment of the presentinvention.

FIG. 2C shows a sample collector in an embodiment of the presentinvention.

FIG. 3A shows a lateral flow test strip in an embodiment of the presentinvention.

FIG. 3B shows a full sandwich including the analyte, the conjugate, andan immobilized binding partner in an embodiment of the presentinvention.

FIG. 3C shows a lateral flow device including the test strip of FIG. 3A,a sample collector, and a sample compressor in an embodiment of thepresent invention.

FIG. 4A shows another lateral flow test strip in an embodiment of thepresent invention.

FIG. 4B shows a full sandwich including the analyte, the conjugate, anda tagged second binding partner in an embodiment of the presentinvention.

FIG. 4C shows a lateral flow device including the test strip of FIG. 4A,a sample collector, and a sample compressor in an embodiment of thepresent invention.

FIG. 5A shows yet another lateral flow test strip in an embodiment ofthe present invention.

FIG. 5B shows a lateral flow device including the test strip of FIG. 5A,a sample collector, and a sample compressor in another embodiment of thepresent invention.

FIG. 6A shows another lateral flow test strip in an embodiment of thepresent invention.

FIG. 6B shows a lateral flow device including the test strip of FIG. 6A,a sample collector, and a sample compressor in another embodiment of thepresent invention.

FIG. 7A shows a device similar to the device of FIG. 3C except that thetest zone is in the sample application zone in an embodiment of thepresent invention.

FIG. 7B shows a device similar to the device of FIG. 4C except that thetest zone is in the sample application zone in an embodiment of thepresent invention.

FIG. 7C shows a device similar to the device of FIG. 5B except that thetest zone is in the sample application zone in an embodiment of thepresent invention.

FIG. 7D shows a device similar to the device of FIG. 6B except that thetest zone is in the sample application zone in an embodiment of thepresent invention.

FIG. 8A shows a lateral flow device in an embodiment of the presentinvention.

FIG. 8B shows another lateral flow device in an embodiment of thepresent invention.

FIG. 9 shows a vertical stack in an embodiment of the present invention.

FIG. 10 shows a prior art gold conjugate sandwich in the test zone.

FIG. 11 shows a sandwich with signal enhancement in the test zone in anembodiment of the present invention.

FIG. 12 shows a sandwich with stacking in the test zone in an embodimentof the present invention.

FIG. 13 shows a schematic exploded view of a lateral flow device withsignal enhancement elements in embodiments of the present invention.

FIG. 14 shows a lateral flow device in another embodiment of the presentinvention.

FIG. 15A shows a stack that forms in an embodiment of the presentinvention.

FIG. 15B shows the stack of FIG. 15A immobilized in the test zone.

FIG. 15C shows a complex that forms in the control zone.

FIG. 16 shows a lateral flow device in another embodiment of the presentinvention.

FIG. 17A shows a stack that forms in an embodiment of the presentinvention.

FIG. 17B shows the stack of FIG. 17A immobilized in the test zone.

FIG. 18 shows a lateral flow device in another embodiment of the presentinvention.

FIG. 19A shows a stack that forms in an embodiment of the presentinvention.

FIG. 19B shows the stack of FIG. 19A immobilized in the test zone.

FIG. 20A shows a lateral flow test strip in an embodiment of the presentinvention.

FIG. 20B shows a “full” sandwich, which preferably forms before reachingthe test line, between the analyte, the labeled conjugate, and a secondtagged mobile binding partner.

FIG. 21A shows another embodiment of a lateral flow test strip withenhancing elements.

FIG. 21B shows the stacked complex at the test line in the presence ofanalyte.

FIG. 21C shows a stacked complex at the test line with additionalenhancing elements.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods and devices for detecting ananalyte (also known as the target) in a sample, where the sample to beanalyzed is applied to a chromatographic carrier. In multi-planarconfigurations for point of care tests, the conjugate containing one ofthe binding partners of the analyte in question is preferably deliveredfrom a different plane. The analyte-containing sample is collecteddirectly from the source and preferably undergoes no prior treatment,elution, dilution, or concentration. The conjugate is made to come incontact with the sample by means of a sample compressor, also referredto herein as a compressor device. Compression aids in combiningmobilized conjugate and sample. The sample compressor, which includesthe conjugate in preferred embodiments, is preferably completelyseparate from the sample analysis device. The sample compressor is notpart of the flow path on the test strip. As a result, the transfer ofthe conjugate and the sample to the sample analysis device, which ispreferably a test strip, is initiated using pressure, not flow orcapillary action. After the sample compressor is applied, if necessarythere may be a time lapse before applying the running buffer. This timelapse between sample application and the initiation of the testing bythe flow can be up to 24 hours or many days depending on the stabilityof the analyte. The non-test strip components, including, depending uponthe embodiment, any combination of the sample compressor, the samplecollector, and one or more external binding partners, preferably remainassociated with the test strip until flow is initiated.

A lateral flow device of the present invention may be an immunoassayusing antibodies or a non-immunoassay using no antibodies but insteadusing other binding partners, including, but not limited to, nucleicacids, nanoparticles, ligands, and receptors.

Before further description of the present invention, and in order thatthe invention may be more readily understood, certain terms have beendefined here as they relate to the present invention:

The term “compression” as used herein refers to the application of thesample and any components on a pad of a sample compressor to the teststrip. The pad, the collection portion of the sample collector, and thesample application zone are all preferably compressible such thatcompression of the three occurs during application of the sample to thetest strip.

The term “pressure” as used herein refers to physical pressure, and morespecifically, physical pressure applied by a sample compressor to asample on a sample collector and, in turn, to a sample application zoneof a test strip. As used herein, pressure, which may be supplied by amechanical bias or a user of the lateral flow device, brings the pad ofthe sample compressor, the collection portion of the sample collector,and the sample application zone of the test strip into physical contactto transfer the sample and any components on the pad of the samplecompressor to the test strip. This transfer preferably does not occur byvertical flow.

The terms “vertical” and “vertically” as used herein refer to thedirection parallel to the thickness or depth, as opposed to the lengthand width dimensions of the elements utilized in the device, such as thepads or mediums.

The terms “lateral” and “laterally” as used herein refer to thedirection parallel to the length, as opposed to the width and depthdimensions of the elements utilized in the device, such as the pads andmediums.

In some embodiments, many of the elements of the test strip aresubstantially planar and have a lateral dimension that is greater thanthe vertical dimension. The magnitudes of these dimensions relative toeach other, however, may be changed within the spirit of the invention.Generally, the terms “vertical”, “vertically”, “lateral”, and“laterally” also refer to the juxtaposition or orientation of theelements of the device. For vertically juxtaposed elements, a linenormal to and intersecting the planar surface of one such element isalso substantially normal to and intersects the planar surface of theother vertically juxtaposed elements.

The term “flow path” as used herein refers to the path of capillary flowin a flow device during use of the device. The flow path in aconventional lateral flow device is laterally along the length of thedevice. In preferred embodiments of the present invention, the flow pathis only lateral, because the sample is vertically transferred bycompression using pressure rather than by flow. In contrast, verticalflow is used to transfer the sample to the test strip in theabove-discussed prior art.

The term “label” as used herein refers to any atom, atoms, molecule, ormolecules, such as a fluorescent tag, used to provide a detectable andpreferably quantifiable signal. Methods of detection of the labelinclude, but are not limited to, visible detection, fluorescence,chemiluminescence, radioactivity, colorimetry, gravimetry, X-raydiffraction, X-ray absorption, magnetism, and enzymatic activity.Visible spectrum test zones may be interpreted by a spectrometer toyield quantified test results.

The term “in situ lysis” as used herein refers to techniques forincorporating lysis agents into a point-of-care testing device, such asa chromatography test strip or other lateral flow immunoassay device, sothat the lysis operation is not conducted as a separate step.

The term “zone” as used herein refers to any portion of the test strip.The boundaries of a zone are preferably planes perpendicular to thelateral direction. The term “zone” also encompasses the term “line”,which refers to a zone having a length in the lateral directionsignificantly smaller than its width.

Embodiments of the present invention include assays where the analyte(target) to be detected does not bind directly to an immobilized bindingpartner in the test zone of a test strip. Instead, the analytepreferably interacts with one or more analyte binding partners in otherzones (or in the buffer, in some embodiments) on the strip. At least oneof the analyte binding partners includes a first tag that forms acomplex with a second immobilized tag in the test zone.

In preferred embodiments, a control zone binding partner is included onthe sample compressor. With this design, if the conjugate zone on thesample compressor is not adequately compressed and made to contact thetest strip, no control zone will develop even with a proper flow of therunning buffer. Thus, the appearance of the control zone with both thenegative and positive test samples indicates a true procedural controlin the test.

In some embodiments of the present invention, when lateral flow begins,the test strip is no longer in compressive contact with the samplecompressor and sample collector. In other embodiments of the presentinvention, however, the vertical stack is maintained during lateral flowto maximize transfer from the sample compressor and sample collector tothe test strip. In yet other embodiments, the sample collector isremoved from the vertical stack after application of the sample to thetest strip, but the sample compressor is then maintained in contact withthe test strip during lateral flow to maximize transfer from the samplecompressor to the test strip.

The invention provides a sensitive and rapid method for the detection ofanalytes, e.g. pathogens, enzymes, immunologic mediators, nucleic acids,proteins, glycoproteins, lipopolysaccharides, protein adducts, tumor andcardiac markers, and/or low-molecular weight compounds, including, butnot limited to, haptens. The methods and devices are suitable fordiagnosis in human beings and animals, e.g. pets or livestock animals.The detection may include direct detection of the analyte and/or thedetection of antibodies against the analyte, which are present in thefluid sample to be tested. Preferably, the method includes a paralleldetermination of a plurality of analytes. The pathogens are preferablyselected from viruses or microorganisms, such as bacteria, fungi (e.g.yeast or molds) or parasites (e.g. amoebae or nematodes). The immunemediators are part of the inflammatory cascade and include, but are notlimited to, antibodies, growth factors, complement, cytokines,lymphokines, chemokines, interferons and interferon derivatives,C-reactive protein, calcitonin, amyloid, adhesion molecules, antibodies,and chemo-attractant components. The low-molecular weight compounds mayinclude drug or chemical molecules or complexes and metabolites formedby drug or chemical molecules.

The detection may include a direct detection of the target, e.g. thepathogen, and/or the detection of antibodies against the target, e.g.the pathogen which are present in the fluid sample to be tested.Preferably, the method includes a parallel determination of a pluralityof targets.

Alternatively, the analyte of interest may be a low-molecular weightcompound. In a preferred embodiment, the analyte to be detected is adrug molecule such as heroin or methamphetamine. In other preferredembodiments, the low-molecular weight compound is a small molecule, suchas a hapten.

The invention also includes the detection of a plurality of pathogens,allergens, immune mediators, nucleic acids, or low-molecular weightcompounds on a single chromatographic carrier. The sample analysisdevice may allow the simultaneous detection of a plurality oflow-molecular weight compounds, immune mediators, nucleic acids,proteins, or pathogens. Although the sample is preferably a fluid,partially or substantially solid dry matter or mass may be tested as asample in devices and methods of the present invention. For example, thefluid may congeal or harden, such as in a healing wound, be collectedwith the sample collector, and then transferred to the sampleapplication zone. The sample may alternatively be a hardened part of ablister scraped from the blister which may be moistened by a body fluidnear the blister site, such as when collecting a sample to be tested fora sexually-transmitted disease, or moistened by the flowing buffer onthe test strip. The sample may be one or more exudates from wounds orblisters.

The body sample is preferably whole blood, serum, plasma, a mucousmembrane fluid (of the oral, nasal, vaginal, anal, inner ear, and ocularcavities), cerebrospinal fluid (CSF), tear fluid, penile fluid, asecretion or exudate from a gland, or a secretion or exudate from alesion or blister, e.g. lesions or blisters on the skin. Morepreferably, the sample is selected from oral, nasal, ocular, genital,and rectal fluids and secretions or exudates from skin lesions orblisters.

In some embodiments, the amount of liquid associated with the sample isinsufficient to transfer the sample and/or any conjugate or secondbinding partner on the pad of the sample compressor to the sampleapplication zone under compression; instead, the running buffer providesthe additional fluid required for transfer of the sample and/orconjugate and/or second binding partner to the sample application zoneof the test strip. In other embodiments, the sample and/or any conjugateor second binding partner on the pad of the sample compressor istransferred to the sample application zone upon compression. Inalternate embodiments, the running buffer may be applied through thecompressor.

In preferred embodiments, the sample is a fluid that does not drip orflow after it is collected. Instead, the fluid is a congealed mass, suchthat, after the sample is collected on the sample collector, the samplecan be held vertically or even upside down, and the sample remains onthe sample collector. For example, when an eye sample is collected andnot subject to pretreatment, the sample remains on the sample collectoreven if held vertically or upside down, primarily due to surfacetension. This is because the sample is effectively trapped and containedon the sample collector material, for example a sample collector fleece.In preferred embodiments, Polyethylene terephthalate (PET) fibers, suchas Dacron® fibers, or nylon fibers are used because the binding is notspecific or permanent, so these fibers “release” the analyte when wet.The phenomenon is similar to gently mopping up a spill by a paper towelsuch that the moisture is held in the pores and by the surface tension.Other materials that could be used for the sample collector fleeceinclude, but are not limited to, polyesters, cellulose, rayon, calciumalginate, microengineered mechanical structures containingmicrocapillaries and/or microchannels, or other fabrics or meshes. Inembodiments where a sterile collector material is needed to collect ahuman body fluid, materials that can be sterilized and are approved forbio-compatibility are preferably used.

A significant advantage of the method is that test results are providedwithin the medical consultation period, e.g. in a few minutes.Preferably, the results are provided in a time period up to 20 minutes,more preferably up to 15 minutes. The test may also be run up to 24 to48 hours after the sample has been taken from the patient. Also, as thetest is noninvasive, it poses very little risk to the patient. Thus, thebest available treatment can be applied on a timely basis for a specificpathogen. A further advantage over prior art methods is that only a fewmicroliters of sample are required to perform an analysis. The sample ispreferably about 0.1 microliter to about 100 microliters, morepreferably about 0.2 microliter to about 20 microliters and mostpreferably about 0.5 microliter to about 15 microliters.

The invention may be performed by means of a simple test kit. Handlingof the test kit does not necessitate additional laboratory equipment,further handling of reagents, or instrumentation. Another importantadvantage of the invention described herein is that the detection limitis typically 10 to 100 times lower than currently available diagnostictests, because samples do not require dilution before they aretransferred to the analysis device. Therefore, the methods of thepresent invention are more sensitive and accurate than methods of theprior art.

If both the conjugate, which includes a first binding partner for theanalyte and a detectable label, and a second binding partner for theanalyte are located on the sample compressor, the sample analysis devicecan be manufactured and used to test for any analyte. The user wouldjust need to choose the specific compressor that contained the bindingpartners that targeted the analyte of interest.

In some of the embodiments of the invention, a body fluid sample isnon-invasively collected with a collection device or swab member. Thecollection step preferably includes wiping or dabbing the swab memberover a surface of the body containing body fluid to be tested.Preferably, the swab member is sterile. The swab member may be dry orpretreated with a fluid before the collection step.

In preferred embodiments, there is no pretreatment of the swab member,and the sample is collected and transferred to the sample analysisdevice without any treatment of the collected sample. By collecting thesample with a collection device and not subjecting the sample topretreatment steps such as extracting and/or diluting the sample,degradation of the sample is avoided. The analyte to be testedpreferably remains intact or in its native form surrounded or mixed withthe other naturally occurring substances in the sample.

In the prior art, when the sample is extracted and diluted in buffer,the sample is often no longer intact. This may change the “conformation”of the analyte due to its stability or lability. By collecting a sampledirectly using a collection device and not pretreating the sample, thenative nature of the sample is preserved in the concentrated form. Sincethis results in a higher concentration of sample in less volume, itincreases the sensitivity of the test. In addition, with no dilution ofthe sample, the time of appearance and the intensity of the test zoneare directly proportional to the analyte concentration. Using aspectrometer, it is possible to get absolute numerical quantification.In addition, not having to pretreat the sample makes the test easier,faster, and less expensive. It also permits the test to be performed ina clinical setting by doctors, nurses, or lab technicians. In teststrips used to detect conjunctivitis, the sensitivity of the tests iscomparable to the sensitivity of ultra-sensitive polymerase chainreaction tests.

The prior art methods and devices required pre-treatment. Some of thereasons that it was believed that pretreatment was necessary includedthe mistaken belief that pretreatment would result in a more homogeneoussample. Another reason was that it was believed that concentratedsamples needed to be buffered before conducting a binding assay. Othersdescribed the need to wash the sample, remove contaminating particlesand substances that potentially could cause a non-specific bindingreaction and therefore a false positive test result. There was also ageneralized belief in the prior art that a larger homogeneous sampleproduced the most sensitive and specific assay test results.

On the contrary, by not pre-treating the sample, the user maintainsinhomogeneous, highly concentrated samples. As described by the materialprinciple of interfacial polarization, in inhomogeneous dielectricmaterials there are charge distributions occurring at the interfaces ofthe phases making up the inhomogeneous dielectric. In an “intact”(undiluted or undisturbed) in vivo infectious body fluid sample thecharges or charge carriers are impeded by trapping at impurity centersor at the phase interfaces. The characteristic of this “intact” sampleresults in a two layer capacitor effect resulting in space-chargepolarization. The characteristic of an “intact” inhomogeneous natureresults in higher binding efficiency and therefore a more sensitiveassay.

It was previously unknown what effects body fluids, including blood,tears, and purulent exudates, would have on different collector fleecematerials. Specifically, it was unknown whether the analytes would beeffectively released from the other cellular material and transferredfrom a sample collector to a sample analysis device.

In some embodiments, the sample size is preferably a few microliters.After transfer of the sample to the sample application zone (preferablywithout treating the sample), elution medium (also known as runningbuffer) is added. Prior art methods of running lateral flow immunoassayswere unable to perform this washing step. For example, when collectingan eye sample to test for eye infections such as conjunctivitis, thesample size is preferably 3 to 15 microliters. In this example, 150 to200 microliters of elution medium is then added to the test strip. As acomparison with different assay systems, this 40 to 50 fold washingexceeds the washing performed in machine dependent ELISA tests.

In one example of collecting a sample, using a gentle swirling motion, asterile swab member may be applied to the body surface or mucousmembrane of concern and allowed to capture any pathogens, low-molecularweight compounds, and/or immune mediators, peptides, glycoproteins,nucleic acids, and allergy-related components contained in the bodyfluid.

The swab member may be a part which is separate from the sample analysisdevice. The sample is then transferred by contacting the swab memberwith the sample analysis device and the sample compressor underconditions, where at least part of the sample is on the swab member. Atleast part of the conjugate in embodiments where the conjugate islocated on the sample compressor and/or at least part of the secondbinding partner in embodiments where the second binding partner islocated on the sample compressor are also transferred to the sampleanalysis device due to pressure. This is a similar phenomenon tosqueezing the fluid out of a sponge. In this embodiment, the swab memberpreferably contacts both a sample application zone on the analysisdevice and the pad portion of the sample compressor (which preferablyincludes the conjugate and/or a second binding partner for the analyte).The sample and conjugate are then transferred to the sample applicationzone and then travel to the detection zone. In some embodiments, theswab member may be fixed in a contact position with the sample analysisdevice in which the sample collection zone of the swab member is indirect contact with the sample application zone of the analysis device.Thus, the swab member and/or the analysis device preferably includesfixing means for providing a fixed contact between both parts in apredetermined position. Alternatively, the swab member may be anintegrated part of the sample analysis device and the transfer includespassing at least a part of the sample on the swab member, as well as theconjugate, to the sample application zone by exerting pressure using thesample compressor. In some embodiments, the sample compressor is also anintegrated part of an integrated sample analysis device and ispreferably connected to the device by a hinge. In other embodiments, thesample compressor is separate from the remainder of the device.

The transfer of the sample from the swab member to the sampleapplication zone on the sample analysis device is preferably a directtransfer, i.e. the transfer takes place without pretreatment of thesample on the swab member. In embodiments without pretreatment of thesample or the swab member, microfiltration occurs in the region wherethe swab member fleece directly contacts the fleece on the strip. Thefibers of the fleece interlock to form a grating or physicalinterference. Thus, larger elements contained in the sample are heldback and not eluted on the sample analysis device. As the conjugate andthe sample move through the sample application zone, the smalleranalytes are eluted. Also, when using samples from mucous membranefluids, mechanical disruption of the mucous in mucous membrane bodilyfluids purifies the sample and the analyte of interest.

In other embodiments, the transfer includes an elution of the samplefrom the swab member with an elution medium, e.g. a buffer or water. Theelution medium may be added from an external source or may be provided,e.g. as a reservoir, within the analysis device. Further, the transferis preferably a chromatographic and/or capillary transfer of fluid tothe detection zone on the sample analysis device.

In some preferred embodiments, the swab member is placed between alateral flow test strip and a pad portion of a sample compressor (whichmay include the conjugate that includes a first binding partner for theanalyte and a detectable label, a second binding partner for the analytethat includes a tag, a control zone binding partner, or any combinationof any of these). With this step, the collected specimen is transferreddirectly onto a test strip. The test strip preferably includes one orseveral capillary active fleeces or membranes.

In some preferred embodiments, the sample is added to a chromatographictest strip, and the conjugate is added as a separate step after thesample is added. In these embodiments, the conjugate and the sample arenot added simultaneously. For example, a sample collector including thesample is placed on a sample application zone of a test strip. At leastsome of the sample is transferred to the test strip at this time. Then,the sample compressor containing the conjugate is added and the samplecompressor compresses the sample collector. This facilitates furthertransfer of the sample, as well as transfer of the conjugate, onto thetest strip. If analyte is present, a complex between the analyte in thesample and the conjugate may be formed as soon as the conjugate beginscompressing the sample. With fluid samples, the complex starts formingdue to the fluid nature of the sample itself. In preferred embodiments,the second binding partner for the analyte is also either on the samplecompressor or in the sample application zone of the test strip. In theseembodiments, the full sandwich between the first binding partner, theanalyte and the second binding partner may be formed before buffer iseven added. Addition of buffer further enhances complex formation andthen transport of the components to the detection zone. Since thecomplex can form during compression, there may be a time lag betweensampling and testing. The reaction between the analyte and the conjugatepreferably begins before buffer is added to the test strip. The time lagbetween when the sample and the conjugate are added and when buffer isadded can be up to 24 hours or even longer.

The detection process will be either started directly with sampletransfer or may require an elution medium to be applied for sampleanalysis. In some embodiments, the elution medium is simple tap water.In other embodiments, the elution medium is an alkaline buffer solution.In the case of an immunochemical test strip where the detection zone islaterally downstream of the sample application zone, the chosen elutionmedium moves towards a detection zone and thereby passes the contactsite within the collection device. The analyte and the conjugate areeluted by the elution medium and carried with it to the detection zone.In the detection zone, the analyte is determined by qualitative and/orquantitative methods, e.g. in an immunological binding reaction.

The test strip can be made of one single chromatographic material, orpreferably several capillary active materials made of the same ordifferent materials and fixed on a carrier backing. These materials arein close contact with each other so as to form a transport path alongwhich a liquid driven by capillary forces flows from the start zone,passing the contact site of the swab and the detection zone, towards awaste zone at the other end of the strip.

Some preferred materials and membranes for the test strip include, butare not limited to, polyethylene terephthalate (PET) fibers, such asDacron® fibers, nitrocellulose, polyester, nylon, cellulose acetate,hydrogel, polypropylene, glass fibers, and combinations of thesematerials and their backings. The characteristics of the fleeces andmembranes depend upon the types of materials used for a particularregion or zone of the test strip or collection device. As describedherein, materials that allow reagents (including those in the reagentzone, the capturing zone, or any of the other zones described herein) tobe mobile and travel with the elution medium include fleece materials orfibers, where the binding is not specific or permanent, so that theanalyte and reagents may be released when they encounter the elutionmedium or with large sample volume. Some of these materials include, butare not limited to, polyethylene terephthalate (PET) fibers, such asDacron® fibers, nylon fibers, polyester fibers, cellulose acetatefibers, polypropylene fibers, glass fibers, foam, sponges, and otherfabrics and meshes. In contrast, materials that immobilize reagents in aparticular zone (including, for example, the reagents immobilized on thetest zone and control zone of the detection zone and the capturingreagents in the embodiments that include capturing reagents immobilizedin a capturing zone downstream of the sample application zone) include,but are not limited to, nitrocellulose and nylon fibers chemicallytreated such that individual fibers in the nylon mesh bind permanentlyto reagents such as proteins. Some methods for manufacturing differentportions of the strip include, but are not limited to, striping,spraying, soaking, and drying materials onto the strip.

While nitrocellulose is used for the detection zone in many of theembodiments of the present invention, in other embodiments, neutralmembranes, such as nylon or polyester may be used. In these embodiments,proteins, such as neutravidin, antibodies and antigens, nanoparticles,or nucleic acids are not immobilized directly. They are insteadconjugated to microspheres which are “deposited” into the membrane andare held in the crevices.

Some preferred materials for the pad portion of the sample compressorinclude, but are not limited to, polyethylene terephthalate (PET)fibers, such as Dacron® fibers, nylon fibers, polyester fibers,cellulose acetate fibers, polypropylene fibers, glass fibers, fleece,foam, sponges, and other fabrics and meshes.

The test strip materials preferably filter and/or retain particulatematter, as well as cell debris, the precipitates, etc., in themembranes. In addition, since the volume of the sample is preferably sosmall, the sample stays put in the materials and the elution bufferflowing directly underneath the sample contacts and transports thesample such that the sample may be extracted, lysed, and/or filteredbefore it reaches the test zone of the detection zone.

Furthermore, devices and test kits of the present invention preferablyperform the methods described herein.

In preferred embodiments, the conjugate is located on a samplecompressor, separate from the sample analysis device. The conjugatepreferably includes a first binding partner for the analyte, as well asbeing labeled with a detectable label. The label is preferablydetectable visibly and/or by fluorescence, but any form of detectionknown in the art may be used, depending upon the label chosen.

In some embodiments, the detectable label for the conjugate can becolloidal gold, colored latex beads, fluorescent nanoparticles,chemiluminiscent nanoparticles, paramagnetic nanoparticles, orphosphorescent nanoparticles.

Qualitative interpretation is performed visually by observing the testzone intensity and hue. In an example where a visual red dye is used asthe label, when the concentration of the analyte is equal or slightlyabove the lower limit of detection, the test zone can be seen faintlyand the hue is pink. As the concentration of the analyte is increased,the test zone intensity correspondingly increases and the hue shiftsfrom pink to bright red. A quantitative interpretation is developedusing a spectrometer operating in the visible spectrum. Either anabsorption measurement or a reflectance measurement may be used in thevisible spectrum to develop the quantification of the test zone. First aset of characterized concentrations of the analyte are developed. Eachof the concentrations is applied to the sample application zone and thetest is run. The spectrometer is used to measure either the absorptionor the reflectance of the test zone. A standard curve is calculated fromthe measured values of the spectrometer. The standard curve is normallylinear. In other embodiments, if fluorescent tags are used, a similarset of known concentrations of the analyte may be developed. An unknownconcentration of the analyte tested and quantified by the spectrometeryields a value that, when plotted on the standard curve, can becorrelated to a concentration of analyte.

The visual label may be any label visible to the naked eye, including,but not limited to, colored particles such as colloidal gold, dyed latexbeads, selenium, or carbon. In some embodiments, the visual tags arealso coated with fluorescing elements. In some embodiments, thefluorescing element is a fluorescing dye. Alternatively, a mixture ofpreferably colorless fluorescing latex bead conjugates is mixed withcolloidal gold (a visible spectrum) conjugates, or conjugates producinga visible read test zone, in lateral flow immunoassays to enhancesensitivity of the assay and to aid in visually reading true positivesand true negatives. In embodiments where nanoparticles are used, thenanoparticles that may be used include, but are not limited to,selenium, carbon, and colloidal gold.

In some embodiments, a second binding partner for the analyte is alsolocated on the sample compressor. The second binding partner includes atag but not a detectable label. The second binding partner mayalternatively be located in the sample application zone of the teststrip, upstream of the sample application zone, or in any location onthe test strip between the sample application zone and the detectionzone. In embodiments where there is a second binding partner for theanalyte either upstream of the detection zone or on the samplecompressor, the detection zone includes an immobile tag that binds tothe tag portion of the second binding partner.

In one preferred embodiment, the second binding partner is tagged withbiotin. In embodiments where the tag on the second binding partner isbiotin, the immobilized tag in the detection zone is preferably avidin,neutravidin, or streptavidin. In other embodiments, the second bindingpartner is tagged with avidin, neutravidin, or streptavidin. In theseembodiments, the immobilized tag in the detection zone is preferablybiotin. Alternatively, the tag on the second binding partner may be alectin and the immobilized tag may be a glycosyl moiety. For example, insome embodiments, the lectin is the Garden pea Lectin and the glycosylmoiety is an erythrocyte glycosyl unit. The tag on the second bindingpartner and the immobilized tag may be reversed within the spirit of thepresent invention. For example, the glycosyl moiety may be the tag onthe second binding partner, with an immobilized lectin tag in thedetection zone. In other embodiments, other receptors and ligands may beused.

In a preferred embodiment, the specific binding partners for theanalytes in the conjugate zone on the sample compressor and/or in thesample application zone are monoclonal, polyclonal, or recombinantantibodies or fragments of antibodies capable of binding to a pathogen.In other embodiments, specific binding partners may also be antigenscapable of binding to antibodies against a pathogen, an immune mediator,peptides, glycoproteins, or an allergen. Other types of binding partnersare bioorganic macromolecules like aptamers or receptors, nanoparticles,or nucleic acids. The methods and devices of the present invention canbe used for any binding assays, and can avoid the use ofantibody/antigens or nucleic acids, for example, in ligand-receptorbinding assays and enzyme-substrate binding assays.

In all of these embodiments, a full “sandwich” is preferably createdbetween the first binding partner of the conjugate, the analyte, and thesecond binding partner, at the sample application zone when the analyteis present. Alternatively, the full “sandwich” may form between thesample application zone and the detection zone, if either of the firstbinding partner or the second binding partner is located downstream ofthe sample application zone. The full sandwich then travels to thedetection zone, where the tag on the second binding partner binds to theimmobilized tag in the detection zone. Note that the complex between thetag on the second binding partner and the immobilized tag in thedetection zone occurs regardless of whether or not the analyte ispresent. However, the complex is only detectable when the analyte ispresent and the conjugate (which includes a detectable label) has boundto the analyte.

In other embodiments, instead of having a second binding partner for theanalyte either on the sample compressor or on the test strip upstream ofthe detection zone, an immobilized second binding partner for theanalyte is located in the detection zone. In these embodiments, half ofthe “sandwich” forms between the first binding partner of the conjugateand the analyte, which then travels to the test zone, where the halfsandwich binds to the immobilized second binding partner, completing thefull “sandwich”.

The device also preferably includes a control zone, which indicateswhether the test was run correctly. In preferred embodiments, a controlzone binding partner, for example a mobile control zone binding partnerwith a visual label, is also located on the sample compressor. Placingthe mobile control zone binding partner, which binds to an immobilizedbinding partner in the control zone, on the sample compressor willindicate whether or not transfer of the conjugate occurred from thesample compressor to the sample application zone of the sample analysisdevice. This is a very useful control, since it is essential that theconjugate be transferred in order to detect the presence of the analyte.

The sample may be taken by a standard swab member as currently used inthe physician's office or emergency rooms. This swab member issubsequently pressed into the sample application zone of thechromatographic test strip using the sample compressor.

In some preferred embodiments, instead of lysing cells “outside” of apoint-of-care testing device, the present invention utilizes “in situlysis”. In these embodiments, the methods and devices of the presentinvention incorporate a lysis zone including at least one lysis agent aspart of a lateral flow assay test strip, such as those discussed herein,or other lateral flow assay devices known in the art, in order to lysethe sample material in situ. In addition, a capturing zone capturesinterfering substances to increase the accuracy of the assay.

Following sample loading, sample traveling with the transport liquidencounters the lysis agent. The lysis agent will have been pre-loadedonto the test strip and is eluted by the transport liquid. In somepreferred embodiments, the lysis agent has been dried into the teststrip. Alternatively, the lysis agent may be pre-dried by freeze dryingor lyophilizing and then pre-loaded into the test strip. In otherembodiments, the lysis agent may be absorbed, adsorbed, embedded, ortrapped on the test strip. In a preferred embodiment, the lysis agent islocalized on the sample application zone or upstream of the sampleapplication zone, so that the sample is lysed when it is transferred tothe sample analysis device. The lysis agent is preferably soluble ormiscible in the sample transport liquid, and the lysis agent issolubilized and activated upon contact with the sample transport liquid.The sample transport liquid then contains both lysis agent in solutionor suspension and sample components in suspension. Any lysis-susceptiblecomponents in the sample, upon being exposed in suspension to the lysisagent, are themselves lysed in situ. The analyte is preferably thenexposed to both the labeled conjugate and the second binding partner, toform the sandwich before reaching the detection zone. Alternatively, thelysis agent may be included in the running buffer.

Alternatively, the lysis agent may be introduced to the test stripduring a sample compression step. In one embodiment, the lysis agent islocated on the pad of the sample compressor. Alternatively, the lysisagent may be dried on the swab member of the sample collector if theswab member does not need to be sterile. Otherwise, the swab member maybe sterilized after addition of the lysis agent using sterilizationtechniques which do not damage the lysing ability of the lysis agent.

The concentration of lysis agent pre-loaded onto a test strip ispreferably between 0.001% and 5% weight/volume. The volume to bepre-loaded depends on where the lysis agent is pre-loaded. Appropriateranges are 1 to 10 microliters when pre-loaded into the sample collectorfleece (the sample application zone) or 5 to 50 microliters whenpre-loaded into the absorbent pad or into other locations within thetest strip. Ideally, the amount pre-loaded should be approximately 3microliters pre-loaded into the sample collector fleece or approximately10 microliters pre-loaded into the absorbent pad or into other locationswithin the test strip.

Selection of a specific lysing environment and agent will depend on theanalyte and the assay. pH and ionic strength are key to the lysingenvironment. As to pH established by the lysis agent, a pH below 4.0tends to precipitate materials, especially proteins. Higher pH, aboveapproximately 10.0, tends to lyse materials such as proteins and cellswalls. Therefore, a pH of approximately 10.0 or above is preferable formany applications. Alternatively, lower pH may be preferred for nucleicacid targets.

As to ionic strength established by the lysis agent, both high and lowionic strength may be used to lyse. For example, a lower ionic strength(hypotonic) tends to break up erythrocytes. Water by itself can lyseerythrocytes. Higher ionic strength environments may be used to rupturecertain cell walls and membranes.

As to specific lysis agents, they may be grouped and selected based ontheir properties: salts, amphoteric and cationic agents, and ionic andnon-ionic detergents. Ammonium chloride (NH₄Cl) lyses erythrocytes.Other salts, including, but not limited to, high concentrations ofsodium chloride (NaCl) and potassium chloride (KCl), may rupture certaincell walls and membranes. Other lysis agents are amphoteric agentsincluding, but not limited to, Lyso PC, CHAPS, and Zwittergent.Alternatively, cationic agents including, but not limited to, C16 TABand benzalkonium chloride may be used as a lysis agent. Both ionic andnon-ionic detergents are often used to break or lyse the cell wall orcell membrane components such as lipoproteins and glycoproteins. Commonionic detergents include, but are not limited to, SDS, EDTA, Cholate,and Deoxycholate. Ionic detergents are good solubilizing agents.Antibodies retain their activity in 0.1% SDS or less. Common non-ionicdetergents include, but are not limited to, Octylglucoside, Digitonin,C12E8, Lubrol, Triton X-100, Noniodet P-40, Tween 20, and Tween 80.Non-ionic and mild ionic detergents are weaker denaturants and often areused to solubilize membrane proteins such as viral surface proteins.Additional lysis agents include, but are not limited to, urea andenzymes. Combinations of different lysis agents may be used to optimizethe lysing environment.

Surfactants generally act as wetting agents and lower the surfacetension of a liquid. This then allows easier spreading by lowering theinterfacial tension between liquids. So, surfactants can interfere withthe natural binding of antigen and antibody or ligand and receptors. Theconcentrations are, therefore, experimentally chosen for each class oflysis agent. Once lysis occurs, it is important that the desired bindingreactions not be hindered. Generally, 0.001% lysis agent concentrationis considered the lower limit, and the upper limit is approximately 1%.There is an additive or synergistic effect when combinations of lysisagents are used. This expands the working range of concentration to runfrom approximately 0.001% to 1%. Finally, some undesirable non-specificbinding may be prevented at a Tween 20 concentration of 5%. In allcases, the total amount of lysis agent pre-loaded onto all locations ofan individual test strip must be sufficient to lyse barriers toimmunodetection, permitting practical operation of the test strip.

The lysis agent itself should not interfere with any other assaydetector or indicator agents and thus does not interfere with any otherassay interactions and reactions to such an extent as to preventpractical operation of the assay. A lysis agent should have sufficientshelf life to allow manufacture, distribution, and storage before use ofa test strip in point-of-care testing.

In a preferred embodiment of the present invention, the lateral flowdevice of the present invention includes a sample-transporting liquid,which can be a buffer, a sample compressor, and a chromatography teststrip containing one or several fleece materials or membranes withcapillary properties through which sample flows. In a device and methodof the invention, it is unnecessary to lyse the cells in the sampleprior to applying the sample to the test strip.

FIG. 1 shows a sample analysis device (test strip) 1 and a samplecollector 2. The sample collector 2 may be any type of sample collector2 known in the art, for example the sample collector 2 could be a swabmember. The sample 20 may include the analyte 3, as well as interferingparticles 5 (which may include interfering proteins or interferinggenes) and other interfering particles or cell debris 4. The sampleanalysis device 1 includes a conjugate zone 8 upstream of the sampleapplication zone 18 in this figure. Although the conjugate zone 8 isshown upstream of the sample application zone 18 in this figure, theconjugate zone 8 may alternatively overlap the sample application zone18 or be downstream of the sample application zone 18 within the spiritof the present invention. The sample application zone 18 is also amicrofiltration zone, which preferably filters out cell debris andinterfering particles 4 that are in the sample 20.

The conjugate zone 8 preferably includes both a mobile conjugate 15,which includes a portion that binds to the analyte 3 and a detectablelabel, and a control zone binding partner 16 with a detectable label,which may be, for example, a control zone antibody with a visual label.In some embodiments, the mobile conjugate is a test antibody conjugatewith a visual label. The control zone binding partner 16 binds with animmobilized binding partner for it in the control zone 11 and indicateswhether the test has run correctly. If the analyte 3 is present in thesample 20, the analyte binds to the conjugate 15, and the conjugate15-analyte 3 complex travel to the test zone 10 in the detection zone12. The analyte 3 then binds to an immobilized binding partner 17 forthe analyte 3, to form the full “sandwich” in a sandwich-type assay.

The transfer of the sample from the sample collector 2 to the sampleapplication zone 18 on the sample analysis device is preferably a directtransfer, i.e. the transfer takes place without pretreatment of thesample on the sample collector 2. In embodiments without pretreatment ofthe sample or the sample collector 2, pressure 14 is applied andmicrofiltration occurs in the region where the sample collector fleecedirectly contacts the fleece on the sample analysis device 1. The fibersof the fleece interlock to form a grating or physical interference.Thus, larger elements contained in the sample, for example cell debrisand interfering particles 4 are held back and not eluted.

The sample application device 1 preferably also includes a blocking zone9 that includes one or more capturing reagents. This blocking zonecaptures interfering proteins and/or genes 5 that may be in the sample20. Capture of an interfering substance 4, 5 by one or more capturingreagents occurs when the capturing reagent interacts in some manner withthe interfering substance to keep the interfering substance frominterfering with the detection of the analyte. While a blocking zone 9is shown in FIG. 1, the capturing reagents may be located in a capturingzone 9 made of materials that allow the capturing reagents to be mobile,in the elution medium, mixed and dried with the reagents, incorporatedinto the sample application zone, incorporated into the sample collectorfleece material, and/or immobilized on an immobilizing material (forexample, nitrocellulose) either as a line or a zone. Any of these or anycombination of these may be used in the embodiments of the presentinvention, depending on the test and sample matrix.

The sample analysis device 1 also optionally includes an absorbent pad 7upstream of the conjugate zone 8 and the sample application zone 18.Buffer is added and travels in the direction of the arrow 6 to elute thetest components, including the sample 20, the conjugate 15, and thecontrol zone binding partner 16, to the detection zone 12. The sampleanalysis device 1 also preferably includes a waste pad 13 at thedownstream end of the device 1. The sample analysis device 1 may alsooptionally include a backing 23.

The devices and methods of the present invention include a samplecompressor 30. Some schematic examples of sample compressors 30 thatcould be used are shown in FIGS. 2A and 2B. The sample compressors 30preferably include a handle 31, an extended portion 32, and a padportion 33. In some designs, the sample compressor includes additionalsections, such as a ledge portion 34 that the pad portion 33 is placedupon. While specific examples are shown in FIGS. 2A and 2B, any samplecompressor 30 that is able to exert pressure to transfer one or morecomponents of the assay and the sample to the sample analysis devicecould be used in the embodiments of the present invention. In preferredembodiments, the conjugate 36 is pre-loaded and dried onto a pad 33 thatforms the conjugate zone. In some preferred embodiments, a labeledcontrol 61 that is able to complex with a binding partner at the controlzone is also pre-loaded and dried onto the pad 33 of the samplecompressor 30. In other preferred embodiments, the second bindingpartner 38 for the analyte is located on the pad 33. Any combination ofthe conjugate 36, the second binding partner 38, or the control zonebinding partner 61 may be on the pad portion 33 of the sample compressor30.

FIG. 2C shows an example of a sample collector 35. In this example, thesample collector 35 is a swab member. The sample collector 35 preferablyincludes a sample collection portion 60, which is preferably made offleece-type materials. In some embodiments, the sample collector 35 issterile.

FIGS. 3A through 3C show one embodiment of a system with a samplecompressor 30, a sample collector 35, and a sample analysis device (atest strip in the figure). The test strip preferably includes anabsorbent pad 42, a sample application zone 44, a detection zone 52, andan optional waste pad 47. The test strip also preferably includes acarrier backing 48. The detection zone 52 preferably includes a testzone 45, which includes an immobilized binding partner 38 for theanalyte 40, as well as a control zone 46. In this embodiment, theconjugate 36 is on the sample compressor 30. The first binding partner37, which is part of the conjugate 36, from the sample compressor 30binds the analyte 40 in the test sample to form a half sandwich, whichis then transported to the second binding partner 38 which isimmobilized in a test zone 45. The full sandwich 420 that forms betweenthe portion 37 of the conjugate 36 that binds to the analyte 40, theanalyte 40, and the second binding partner 38 is shown in FIG. 3B. Inpreferred embodiments, the pad 33 on the sample compressor 30 alsoincludes a control zone binding partner 61 with a detectable label. Thecontrol zone binding partner 61 complexes with its binding partner inthe control zone 46. Including the control zone binding partner 61 onthe sample compressor 30, instead of on the test strip or in the bufferas known in the prior art, permits the user to be sure that thecomponents on the sample compressor 30, which, in this embodimentinclude both the conjugate 36 and the control zone binding partner 61,have effectively transferred to the sample analysis device and thusensures proper operation of the system.

In one example, both the first binding partner 37 and the second bindingpartner 38 are different antibodies to the analyte. The control zonebinding partner 61 is also preferably an antibody, and its bindingpartner at the control zone is an antigen (or vice versa). In otherembodiments, specific binding partners may also be antigens capable ofbinding to antibodies against the analyte. Other types of bindingpartners are bioorganic macromolecules like aptamers or receptors,nanoparticles, or nucleic acids. The device shown in FIGS. 3A-3C of thepresent invention can be used for any binding assays, and can avoid theuse of antibody/antigens or nucleic acids, for example, inligand-receptor binding assays and enzyme-substrate binding assays.

In operation, the sample collector 35 is placed such that the sample isdirectly above the sample application zone 44. In some embodiments,placement of the sample collector 35 above the sample application zone44 is not simultaneous with placement of the sample compressor 30. Inother words, in these embodiments, some of the sample is transferred tothe sample application zone 44 before the sample compressor 30 is addedto the vertical stack.

The sample compressor 30 exerts pressure 51 on the sample collector 35,using pressure to transfer the sample, including the analyte 40 (ifpresent), and the conjugate 36 onto the sample application zone 44. Ifthere is also a control zone binding partner 61 on the sample compressor30, the control zone binding partner 61 is also transferred. Note thatthe transfer is due to pressure, not due to flow or capillary action.Then, buffer 43 is added to permit flow of the conjugate 36-analyte 40complex (if present) to the detection zone 52. An immobilized bindingpartner 38 in the test zone 45 then binds the analyte, forming thecomplete sandwich. Since the conjugate 36 includes a label 41, thecomplex that forms is detectable and indicates a positive result. Properoperation of the test also results in a detectable positive result inthe control zone 46 due to the interaction between the control zonebinding partner 61 and its immobilized partner in the control zone 46.

Although it is not shown, there may also optionally be a lysis zone,which preferably overlaps or is upstream of the sample application zone44. In other embodiments, there may be a blocking zone that includescapturing reagents, similar to the zone discussed with respect to FIG.1.

In other embodiments, the conjugate zone can contain both the bindingpartners for the analyte in the sample to form a “full sandwich”. One ofthe binding partners preferably has a suitable marker such as biotin,avidin, lectin, a glycosyl moiety, a specific ligand, or a specificreceptor. The other can be conjugated to the appropriate nanoparticlesas mentioned below. The full sandwich is then captured at the test zonewhere the binding partner of the suitable marker, including, but notlimited to, avidin for biotin, biotin for avidin, glycosyl moiety forlectin, lectin for the glycosyl moiety, a receptor for the ligand, or aligand for the receptor, is immobilized.

FIG. 20A shows an example of a test strip in an embodiment of thepresent invention. The test strip preferably includes an absorbent pad42, a sample application zone 44, a detection zone 52, and an optionalwaste pad 47. The test strip also preferably includes a carrier backing48. In this embodiment, the entire sandwich (first binding partner513-analyte-40-second binding partner-518) forms in the sampleapplication zone 44. The “full sandwich” 514 is shown in FIG. 20B. Thetest zone 45 in this embodiment includes an immobilized tag 510 thatbinds to the tag 519 of the second binding partner 518. The immobilizedtag 510 does not bind directly to the analyte 40; instead, it bindsthrough an intermediary, the tag 519 on the second binding partner 518for the analyte 40.

In this embodiment, a first binding partner 513, which is part of thelabeled conjugate 505, binds the analyte 40 in the test sample to formhalf a sandwich. The second binding partner 518 also includes a tag 519.The second binding partner 518 in this embodiment is preferablypre-loaded and dried on the sample application zone 44 of the teststrip, while the labeled conjugate 505 is preferably pre-loaded anddried onto a labeled conjugate zone 515 upstream of the sampleapplication zone 44. Alternatively, the second binding partner 518and/or the labeled conjugate zone 515 may be located anywhere on thetest strip upstream of the detection zone 52 including, but not limitedto, overlapping the sample application zone 44, upstream of the sampleapplication zone 44, or between the sample application zone 44 and thedetection zone 52. In one preferred embodiment, approximately 75-80% ofthe labeled 509 conjugate 505 is upstream of the sample application zone(with approximately 20-25% of the labeled conjugate 505 overlapping thesample application zone 44) and approximately 75-80% of the secondbinding partner 518 is located downstream of the sample application zone44 (with approximately 20-25% of the second binding partner overlappingthe sample application zone 44). Although not preferred, in otherembodiments, either the labeled conjugate 505, the second bindingpartner 518, or both may be located in the buffer or pre-mixed with thesample before the sample is added to the test strip. In still otherembodiments, any or all of the components could overlap the detectionzone 52.

In some embodiments, both the first binding partner 513 and the secondbinding partner 518 are different antibodies to the analyte 40. In otherembodiments, specific binding partners may also be antigens capable ofbinding to antibodies against the analyte. Other types of bindingpartners are bioorganic macromolecules like aptamers or receptors,nanoparticles or nucleic acids. The device shown in FIG. 20A can be usedfor any binding assays, and can avoid the use of antibody/antigens ornucleic acids, for example, in ligand-receptor binding assays and enzymesubstrate binding assays.

In one preferred embodiment, the second binding partner 518 is tagged519 with biotin. In embodiments where the tag 519 on the second bindingpartner 518 is biotin, the immobilized tag 510 in the detection zone 52is preferably avidin, neutravidin, or streptavidin. In otherembodiments, the second binding partner 518 is tagged 519 with avidin,neutravidin, or streptavidin. In these embodiments, the immobilized tag510 in the detection zone 52 is preferably biotin. Alternatively, thetag 519 on the second binding partner 518 may be a lectin and theimmobilized tag 510 may be a glycosyl moiety. For example, in someembodiments, the lectin is the Garden pea Lectin and the glycosyl moietyis an erythrocyte glycosyl unit. The tag on the second binding partnerand the immobilized tag may be reversed within the spirit of the presentinvention. For example, the glycosyl moiety may be the tag on the secondbinding partner, with an immobilized lectin tag in the detection zone.In other embodiments, other receptors and ligands may be used for thetags.

In operation, a sample collector containing the sample is placed suchthat the sample is directly above the sample application zone 44. Inpreferred embodiments, the sample has not been subject to pretreatmentprior to application to the test strip. Instead, the sample is still inits native form.

The sample is transferred to the sample application zone 44 of the teststrip. A sandwich forms with the labeled conjugate 505 as one piece ofbread and the second binding partner 518 as a second piece of bread,with the analyte 40 in between them, when the three components come intocontact with each other during flow 43. The labeled conjugate505-analyte 40 (if present)-second binding partner 518 complex (acomplete sandwich) flow to the detection zone 52. An immobilized tag 510in the test zone 45 then binds the tag 519. Since the labeled conjugate505 includes a label 509, the complex that forms is detectable andindicates a positive result. Proper operation of the test also resultsin a detectable positive result in the control zone 46, preferably dueto the interaction between a control line binding partner and itsimmobilized partner in the control zone 46.

Although it is not shown, there may also optionally be a lysis zone,which preferably overlaps the sample application zone 44 or isalternatively located in other portions of the test strip within thespirit of the present invention.

In some preferred embodiments using tags, the detection zone includes anantibody against the tag. The antibody may be a monoclonal, polyclonalor single domain antibody. For example, when the tag is biotin, ananti-biotin antibody is immobilized in the test zone instead of avidin,neutravidin, or streptavidin.

FIGS. 4A through 4C show an example of an embodiment of the system witha sample compressor 30, a sample collector 35, and a sample analysisdevice (a test strip in the figure). Similar to FIG. 3A-3C, the teststrip preferably includes an absorbent pad 42, a sample application zone44, a detection zone 52, and an optional waste pad 47. The test stripalso preferably includes a carrier backing 48. In this embodiment, theentire sandwich (first binding partner 37-analyte-40-second bindingpartner-38) forms in the sample application zone 44 (preferably beforethe addition of buffer). In some embodiments, placement of the samplecollector 35 above the sample application zone 44 is not simultaneouswith placement of the sample compressor 30. In other words, in theseembodiments, some of the sample is transferred to the sample applicationzone 44 before the sample compressor 30 is added to the vertical stack.

The test zone 45 in this embodiment includes an immobilized tag 50 thatbinds to the tag 39 of the second binding partner 38. In thisembodiment, a first binding partner 37, which is part of the conjugate36 and is preferably pre-loaded and dried on the pad 33 of the samplecompressor 30, binds the analyte 40 in the test sample to form a halfsandwich. The second binding partner 38 in this embodiment is alsopreferably pre-loaded and dried on the pad 33 of the sample compressor.The second binding partner 38 also includes a tag 39.

The full sandwich 420 that forms between the binding partner 37 of theconjugate 36, the analyte 40, and the second binding partner 38 in thisembodiment (as well as the embodiments in FIGS. 5A-5B, 6A-6B, 7B, 7C,and 7D) is shown in FIG. 4B. In preferred embodiments, the pad 33 on thesample compressor 30 also includes a control zone binding partner 61(shown in FIG. 3C) with a detectable label. The control zone bindingpartner 61 complexes with its binding partner in the control zone 46.Including the control zone binding partner 61 on the sample compressor30, instead of on the test strip or in the buffer as known in the priorart, permits the user to be sure that the components on the samplecompressor 30, which include both the conjugate 61 and the control zonebinding partner 61, have effectively transferred to the sample analysisdevice and thus ensures proper operation of the system.

In one example, both the first binding partner 37 and the second bindingpartner 38 are different antibodies to the analyte. The control zonebinding partner 61 is also preferably an antibody, and its bindingpartner at the control zone is an antigen (or vice versa). In otherembodiments, specific binding partners may also be antigens capable ofbinding to antibodies against the analyte. Other types of bindingpartners are bioorganic macromolecules like aptamers or receptors,nanoparticles, or nucleic acids. The device shown in FIGS. 4A-4C of thepresent invention can be used for any binding assays, and can avoid theuse of antibody/antigens or nucleic acids, for example, inligand-receptor binding assays and enzyme-substrate binding assays.

In one preferred embodiment, the second binding partner 38 is taggedwith biotin 39. In embodiments where the tag 39 on the second bindingpartner 38 is biotin, the immobilized tag 50 in the detection zone ispreferably avidin, neutravidin, or streptavidin. In other embodiments,the second binding partner 38 is tagged 39 with avidin, neutravidin, orstreptavidin. In these embodiments, the immobilized tag 50 in thedetection zone 52 is preferably biotin. Alternatively, the tag 39 on thesecond binding partner 38 may be a lectin and the immobilized tag 50 maybe a glycosyl moiety. For example, in some embodiments, the lectin isthe Garden pea Lectin and the glycosyl moiety is an erythrocyte glycosylunit. The tag on the second binding partner and the immobilized tag maybe reversed within the spirit of the present invention. For example, theglycosyl moiety may be the tag on the second binding partner, with animmobilized lectin tag in the detection zone. In other embodiments,other receptors and ligands may be used for the tags.

In operation, the sample collector 35 is placed such that the sample isdirectly above the sample application zone 44. The sample compressor 30exerts pressure 51 on the sample collector 35. The pressure transfersthe sample (including the analyte 40, if present), the conjugate 36, andthe tagged second binding partner 38 onto the sample application zone44. If there is also a control zone binding partner 61 on the samplecompressor 30, the control zone binding partner 61 is also transferred.Note that the transfer is due to pressure, not due to flow or capillaryaction. Then, buffer 43 is added to permit flow of the conjugate36-analyte 40 (if present)-second binding partner 38 complex (a completesandwich) to the detection zone 52. An immobilized tag 50 in the testzone 45 then binds the tag 39. Since the conjugate 36 includes a label41, the complex that forms is detectable and indicates a positiveresult. Proper operation of the test also results in a detectablepositive result in the control zone 46 due to the interaction betweenthe control zone binding partner 61 and its immobilized partner in thecontrol zone 46.

Although it is not shown, there may also optionally be a lysis zone,which preferably overlaps the sample application zone 44. In otherembodiments, there may be a blocking zone that includes capturingreagents, similar to the zone discussed with respect to FIG. 1.

In another embodiment, the two binding partners for the analyte arelocated in such a way to achieve a “vertical sandwich” where the samplebinds with the conjugate being compressed from the second plane and canbind simultaneously or concurrently with the other binding partnerlocated on the strip in the plane of the strip. Thus a sandwiching ofthe analyte in the sample is achieved by binding to the partner from theconjugate delivered from above the plane of the strip and binding to thesecond binding partner located on the plane of the strip below thesample delivering material.

FIGS. 5A and 5B show another example of an embodiment of the system witha sample compressor 30, a sample collector 35, and a sample analysisdevice (a test strip in the figure). Similar to FIG. 3A-3C, the teststrip preferably includes an absorbent pad 42, a sample application zone44, a detection zone 52, and an optional waste pad 47. The test stripalso preferably includes a carrier backing 48. Similar to the embodimentshown in FIGS. 4A and 4C, in this embodiment, the entire sandwich (firstbinding partner 37-analyte 40-second binding partner 38) forms in thesample application zone 44. The test zone 45 in this embodiment includesan immobilized tag 50 that binds to the tag 39 of the second bindingpartner 38. In this embodiment, a first binding partner 37, which ispart of the conjugate 36 and is preferably pre-loaded and dried on thepad 33 of the sample compressor 30, binds the analyte 40 in the testsample to form a half sandwich. The second binding partner 38 in thisembodiment is preferably pre-loaded and dried on the sample applicationzone 44 of the test strip. The second binding partner 38 also includes atag 39. Alternatively, the second binding partner 38 in this embodimentmay be located anywhere on the test strip upstream of the detection zoneincluding, but not limited to, overlapping the sample application zone,upstream of the sample application zone, and between the sampleapplication zone and the detection zone.

In preferred embodiments, the pad 33 on the sample compressor 30 alsoincludes a control zone binding partner 61 (shown in FIG. 3C) with adetectable label. The control zone binding partner 61 complexes with itsbinding partner in the control zone 46. Including the control zonebinding partner 61 on the sample compressor 30, instead of on the teststrip or in the buffer as known in the prior art, permits the user to besure that the components on the sample compressor 30, which include boththe conjugate 61 and the control zone binding partner 61, haveeffectively transferred to the sample analysis device and thus ensuresproper operation of the system.

In one example, both the first binding partner 37 and the second bindingpartner 38 are different antibodies to the analyte. The control zonebinding partner 61 is also preferably an antibody, and its bindingpartner at the control zone is an antigen (or vice versa). In otherembodiments, specific binding partners may also be antigens capable ofbinding to antibodies against the analyte. Other types of bindingpartners are bioorganic macromolecules like aptamers or receptors,nanoparticles, or nucleic acids. The device shown in FIGS. 5A-5B of thepresent invention can be used for any binding assays, and can avoid theuse of antibody/antigens or nucleic acids, for example, inligand-receptor binding assays and enzyme-substrate binding assays.

In one preferred embodiment, the second binding partner 38 is taggedwith biotin 39. In embodiments where the tag 39 on the second bindingpartner 38 is biotin, the immobilized tag 50 in the detection zone ispreferably avidin, neutravidin, or streptavidin. In other embodiments,the second binding partner 38 is tagged 39 with avidin, neutravidin, orstreptavidin. In these embodiments, the immobilized tag 50 in thedetection zone 52 is preferably biotin. Alternatively, the tag 39 on thesecond binding partner 38 may be a lectin and the immobilized tag 50 maybe a glycosyl moiety. For example, in some embodiments, the lectin isthe Garden pea Lectin and the glycosyl moiety is an erythrocyte glycosylunit. The tag on the second binding partner and the immobilized tag maybe reversed within the spirit of the present invention. For example, theglycosyl moiety may be the tag on the second binding partner, with animmobilized lectin tag in the detection zone. In other embodiments,other receptors and ligands may be used for the tags.

In operation, the sample collector 35 is placed such that the sample isdirectly above the sample application zone 44. The sample compressor 30exerts pressure 51 on the sample collector 35, using pressure totransfer the sample (including the analyte 40, if present) and theconjugate 36 onto the sample application zone 44. A “vertical” sandwichforms with the conjugate 36 as the top piece and the second bindingpartner 38 as the bottom piece, with the analyte 40 in between them. Ifthere is also a control zone binding partner 61 on the sample compressor30, the control zone binding partner 61 is also transferred. Note thatthe transfer is due to pressure, not due to flow or capillary action.Then, buffer 43 is added to permit flow of the conjugate 36-analyte 40(if present)-second binding partner 38 complex (a complete sandwich) tothe detection zone 52. An immobilized tag 50 in the test zone 45 thenbinds the tag 39. Since the conjugate 36 includes a label 41, thecomplex that forms is detectable and indicates a positive result. Properoperation of the test also results in a detectable positive result inthe control zone 46 due to the interaction between the control zonebinding partner 61 and its immobilized partner in the control zone 46.

Although it is not shown, there may also optionally be a lysis zone,which preferably overlaps or is located upstream of the sampleapplication zone 44. In other embodiments, there may be a blocking zonethat includes capturing reagents, similar to the zone discussed withrespect to FIG. 1.

FIGS. 6A and 6B show another embodiment of the present invention, wherethe sample compressor 30 includes the second binding partner 38 for theanalyte 40, coupled with a tag 39, and the test strip includes theconjugate 36, which includes both a first binding partner 37 for theanalyte 40 and a detectable label 41, and the immobilized tag 50 thatbinds to the tag on the second binding partner in the test zone 45. Thisembodiment operates similarly to the embodiment described with respectto FIGS. 5A and 5B, except that the “vertical” sandwich forms with thesecond binding partner 38 as the top piece and the conjugate 36 as thebottom piece, with the analyte 40 in between them. Alternatively, theconjugate 36 in this embodiment may be located anywhere on the teststrip upstream of the detection zone including, but not limited to,overlapping the sample application zone, upstream of the sampleapplication zone, or between the sample application zone and thedetection zone.

FIGS. 7A through 7D are similar to FIGS. 3C, 4C, 5B, and 6B,respectively, except that the detection zone 52 overlaps the sampleapplication zone 44 in these figures. The detection zone in theseembodiments is preferably made of nitrocellulose. Although no lateralflow is strictly required to run the assay in these embodiments, atleast a nominal amount of flow is preferred such that the sandwich isable to bind in the test zone and any unbound conjugate is washed out ofthe test zone. In one embodiment, instead of a running buffer beingapplied to an end of the test strip, a washing fluid may be applieddirectly to the test zone, either from above or from the side, forexample using a water bottle. In one embodiment, the sample compressorand the sample collector are substantially transparent so that the testzone can be read without removal of the vertical stack from the teststrip. Note that, while both the test zone 45 and the control 46 areshown within the sample application zone in these figures, in otherembodiments the test zone 45 could overlap the sample application zone44 while the control zone 46 is downstream of the sample applicationzone 44. If the control zone was laterally downstream from the sampleapplication zone 44, it would be necessary to add buffer to allow flow.In addition, it may be preferable to add a buffer, for example a bufferthat includes silver, to enhance the signal from a positive result.

A universal test strip 80, as shown in FIG. 8A, may be used when thesample compressor 30 includes both of the binding partners 37, 38 forthe analyte 40. The sample compressor 30 and the sample collector 35would be transferred to the universal test strip 80 at the sample window81. Since the elements specific to the analyte 40 being tested are onthe sample compressor 30, the test zone 83 in the viewing window 82 ofthe universal test strip 80 only needs to have a tag 50 that complexeswith the tag 39 on the second binding partner 38 for the analyte 40. Forexample, when the second binding partner 38 for the analyte 40 is tagged39 with biotin, the test zone 83 of the universal test strip 80 wouldinclude avidin 39, a binding partner for biotin. The universal teststrip 80 also preferably includes a control zone 84 and a housing 85.For the embodiments of FIGS. 7A through 7D, the test zone is located inthe sample window 81. In other embodiments, the suitable marker can be anucleotide sequence that can hybridize with the suitable nucleic acidsequence immobilized at the test zone.

Although the sample compressor and the sample collector are shown asseparate entities in FIGS. 1-8A, the pad 33 of the sample compressor andthe sample collector portion 60 of the sample collector may becomponents of a single element within the spirit of the presentinvention. For example, the sample collector may be rotatably orflexibly or connected as part of a cartridge to the sample compressor,such that a sample can be collected from a patient with the samplecollection portion without exposing the patient to the sample compressorpad and then the sample collection portion and sample compressor pad canbe brought into contact for application to the sample application zoneof the test strip by compression. The sample collector also may berotatably or flexibly connected to the test cassette or may be insertedas a cartridge. In another embodiment, the sample may be forciblyinjected directly onto the test strip prior to placing the compressorand/or conjugates into position. In yet another embodiment, the samplecollector may contact the conjugates in an external cartridge that thensnaps or inserts into a test cassette to bring the material in contactwith the test strip.

In some embodiments, the sample compressor 30 is rotatably connected tothe housing 85 as shown in FIG. 8B. While the hinge of the samplecompressor 30 is shown such that the sample compressor 30 is rotatedtowards the downstream end of the strip when open, the housing could bedesigned such that the sample compressor 30 is hinged to either side orin other directions within the spirit of the present invention. Thesample collection portion 60 of the sample collector 35 is preferablyinserted from the side such that it lines up with an insertion hole 88on the side of the housing 85. However, the sample collector 35 could beinserted in any direction depending upon the design of the housing. Thesample compressor 30 preferably includes a pad (not visible in FIG. 8B),with one or more assay components, located on the surface of the samplecompressor facing the sample application zone of the test strip 80. Thesample compressor 30 is then closed such that a compression pressure isapplied to the vertical stack of the pad of the sample compressor, thesample collection portion, and the sample application zone to transferthe sample and the one or more assay components to the sampleapplication zone of the test strip. While there is an absorbent padsticking out of the housing at the far upstream end of the device inFIG. 8B, the length of the absorbent pad may vary. In fact, as long asbuffer can be added at the upstream end (for example, through anapplication window in the housing), it is not necessary to have theabsorbent pad extend significantly outside the housing. In thisembodiment, there is no possibility of losing the sample compressor, andthere is no need to align the sample compressor with the sampleapplication zone when forming the vertical stack. One advantage of theseembodiments is that they allow for a time lapse between sampleapplication and the actual initiation of flow to the test zone. In otherwords, the sandwich can be pre-made, and the flow initiated much later.

Alternatively, the pad 33 may be separate from the sample compressorwithin the spirit of the present invention. The pad may be on a bindingpartner applicator similar to the sample collector. In theseembodiments, the binding partner applicator may be located between thesample collection portion and the sample application zone when thepressure is applied by the sample compressor to transfer the sample tothe sample application zone.

FIG. 9 shows a vertical stack including a sample compressor 30, a samplecollector 35 with a sample collection portion 60, a binding partnerapplicator 62 with an applicator pad 64, and a sample application zone44 of a test strip. While the binding partner applicator 62 includes ahandle in FIG. 9, the binding partner applicator 62 could alternativelysimply be a pad. The ledge portion 34 of the sample compressor 30applies pressure to the sample collection portion 60 loaded with asample and the applicator pad 64 loaded with at least one bindingpartner for an analyte to be tested for in the sample. The pressurepreferably forces at least a portion of the sample from the samplecollection portion 60 to wet the applicator pad 64, thereby mobilizingsome of the binding partner such that at least some of the sample andsome of the binding partner are transferred to the sample applicationzone 44. In some embodiments, this transfer occurs without dilution. Inembodiments with small sample volumes or viscous or solid samples,however, an additional liquid may be used to facilitate transfer of thesample and the binding partner to the test strip. In some embodiments,as shown in FIG. 9, the sample compressor has no pad, although a pad maybe used to aid in transfer, such as by supplying additional liquid orbuffer, within the spirit of the present invention. In some embodiments,as shown in FIG. 9, the sample collection portion 60 is located betweenthe sample compressor 30 and the applicator pad 64 in the vertical stackto aid in transfer of the binding partner to the test strip duringcompression. Alternatively, the applicator pad 64 may be placed betweenthe sample compressor 30 and the sample collection portion 60 within thespirit of the present invention. In embodiments where the full sandwichforms prior to reaching the test zone, two binding partner applicators(a separate applicator for each binding partner of the analyte) may beused, with the sample collection portion, the first applicator pad, andthe second applicator pad being placed in any order on the verticalstack within the spirit of the present invention. Alternatively, asingle binding partner applicator could include both of the bindingpartners for the analyte. In other embodiments, the sample, the firstbinding partner, and the second binding partner may be appliedsequentially to the test strip in any order using the sample compressorwithin the spirit of the present invention.

In a method of applying a sample to a test strip of a lateral flowdevice, at least one external binding partner is first placed on thesample application zone of the test strip. The external binding partnermay be located on an external pad. In embodiments where there are twoanalyte binding partners that bind the analyte prior to reaching thetest zone, either one or both of the analyte binding partners may beadded. A sample collector that includes the sample is placed in avertical stack between the external binding partner and a samplecompressor. The sample compressor applies pressure to the samplecollector to transfer the external binding partner and at least aportion of the sample to the sample application zone. Alternatively, theexternal binding partner could be added and compressed by the samplecompressor, then removed, before the sample collector is stacked abovethe sample application zone, where the sample is compressed onto thetest strip. In another alternative embodiment, at least one externalbinding partner is placed in the vertical stack between the samplecompressor and sample collector. Alternatively, the sample collector isadded and compressed, then removed, and then the external bindingpartner is added and compressed onto the test strip. In otherembodiments, the sample collector is in a vertical stack between a firstexternal binding partner and a second external binding partner, and thesample compressor applies pressure to the vertical stack. In theseembodiments, neither the strip nor the sample compressor has a specificanalyte binding partner. The sample, the analyte binding partner, andthe mobile control binding partner may also be applied to the sampleapplication zone in multiple steps in any combination within the spiritof the present invention.

Alternatively, in a lateral flow device of the present invention, thesample compressor may be a universal sample compressor with nocomponents specific to the analyte of interest. In one embodiment, thesample compressor contains no components of the assay. In embodimentswith a control, the pad of the sample compressor contains only themobile control zone binding partner. In these embodiments, one or morebinding partner applicators include at least one binding partner for theanalyte and become part of the vertical stack with the sample compressorand the sample collector when the sample is transferred to the sampleapplication zone. The sample, the analyte binding partner, and themobile control binding partner may also be applied to the sampleapplication zone in multiple steps in any combination within the spiritof the present invention.

In another embodiment of the present invention, the sample compressor 30also serves as the sample collector, and the pad 33 of the samplecompressor also serves as the sample collection portion. In thisembodiment, the conjugate, the second binding partner, the control linebinding partner, and/or any combination of the three, are preferablylocated on a back surface of the pad 33, where the pad is attached tothe sample compressor arm. In embodiments where sample collection needsto be performed sterilely, the sample compressor 30 is then preferablysterilized by radiation prior to use as a sample collector. The sampleis then collected using the front part of the pad so that the patient isnot exposed to the conjugate or the second binding partner during sampleacquisition. When the sample is applied to the sample application zoneof the test strip, the pad is preferably compressed so that the samplemixes with the conjugate or the second binding partner and at least aportion of both is squeezed out onto the test strip.

In some embodiments, a lateral flow device of the present invention mayalso include a built-in, on-line, or in situ signal amplificationsystem. The sample amplification system may be used in combination witha sample compressor or in a method or device without a sample compressorwithin the spirit of the present invention. In embodiments wherecolloidal gold is used as the detectable label for the conjugate, thesignal of the colloidal gold in the conjugate bound to the test zone canbe further amplified by silver enhancement. Suitable formulations ofsilver salts and the silver developers can be dried at the site ofsample application or upstream to it or downstream to it. The silversalts and the developers can be dried together, upstream or downstreamto each other, or can be separated by the sample application area. Inother embodiments, stacking, where the system includes a conjugate withan additional antigen and a second conjugate, which is preferably ananoparticle, with the specific binding partner of the antigen, is usedto amplify the signal. The second conjugate also preferably includes alabel. In the second conjugate, the binding partner may be conjugated toa particle that is the same size, smaller, or larger size than theparticle in the first conjugate. In yet other embodiments, both thesilver enhancement and the stacking enhancement may be used on the sametest strip. The stacking conjugate and silver enhancement elements canbe together or upstream or downstream to one another. A preferredfeature of these embodiments is that both the “stacking” nanoparticlesand/or silver enhancers do not come into contact with the conjugateinitially but come into contact only while the conjugate is immobilizedat the test zone. Thus, a better specificity is achieved.

In some embodiments where a “full sandwich” is formed between theanalyte 40, the first analyte binding partner 37, and the second analytebinding partner 38 prior to the complex reaching the detection zone 52(see, for example, FIGS. 4A-4C, 5A-5B, and 6A-6B), silver enhancement orother amplification signals may be placed upstream of the sampleapplication zone 44 such that the silver salt and/or silver developerinteracts with the full sandwich before the complex reaches thedetection zone 52. In other embodiments with a full sandwich, the silversalt and/or silver developer are located downstream of the sampleapplication zone 44 such that the full sandwich forms and travels to thesilver salt/developer before reaching the detection zone 52.

In the prior art as shown in FIG. 10, there is a one-to-onecorrespondence between analyte 40 and label 41 at the test zone 45,because each analyte binds to one immobilized binding partner 38 and onemobile binding partner 37 with one label 41 on the conjugate 36.

In a signal amplification system of the present invention, theamplification source may be located anywhere on the test strip,including at the sample application zone, or upstream or downstream ofit. Alternatively, the source of amplification may be located in thebuffer or on the sample compressor.

In some embodiments as shown in FIG. 11, the amplification source 70non-specifically deposits itself onto the conjugate such that multipleconjugates are associated with one analyte bound in the test zone. Inthis embodiment, the amplification source is preferably one or moresilver salts, and a silver developer may be used to enhance the signalin assays using colloidal gold as the label portion 41 of the conjugate.The silver salts and silver developer may be located or introduced inany manner to enhance detection of the analyte. In embodiments wherecolloidal gold is used as the detectable label for the conjugate, thesignal of the colloidal gold in the conjugate bound in the test zone canbe amplified by silver enhancement. Suitable formulations of silversalts and the silver developers can be dried at the site of sampleapplication or upstream to it or downstream to it. Silver salts and thedevelopers can be dried together, upstream or downstream to each other,or separated by the sample application area. Alternatively, silver saltsand/or developers can be included as part of the buffer.

In a preferred embodiment, the mixture of the silver salts anddevelopers is dried in an area between the sample application zone andthe test zone. In this embodiment, a full sandwich of the analytebetween two binding partners (one being a conjugate on gold and theother suitably tagged with markers such as biotin) moves into the silverenhancing area and together travel to the test zone where they getcaptured. Although the silver enhancement may be applied to the halfsandwich prior to capture, the silver enhancement is preferably appliedafter capture, because it may otherwise interfere with binding at thetest zone. Silver salts and developers may be used in any of theembodiments described herein, including, but not limited to those shownin FIGS. 3A-3C, 4A-4C, 5A-5B, 6A-6B, and 7A-7D.

In yet another embodiment, the silver enhancing area is located directlyunderneath the sample application material. The compressor with both thebinding partners as described above would form the full sandwich andbecome enhanced by silver salts and developers all in one place. Thismega complex then can move into the test zone where it can be captured.

In yet another embodiment, the silver enhancement is achieved byincorporating the silver salts and the developers in the running buffer.In other embodiments, the silver salts and/or silver developer may belocated on the sample compressor or the sample collector in situationswhere the sample collector need not be sterile. Otherwise, the samplecollector may be sterilized after addition of the silver salts and/orsilver developer using sterilization techniques, such as, for example,radiation, which do not damage the silver salts and/or silver developer.

In yet another embodiment, the silver salt are dried at the site,upstream, or downstream to the sample application area and the silverdeveloper can be added to the viewing window as a separate step.

In a preferred embodiment involving the silver enhancement, since silveris light-sensitive, the test is run upside down (with the cassetteturned over in embodiments where a cassette is used) or otherwiseshielded from ambient light prior to the completion of the test.

In another embodiment, the silver enhancement is achieved as a separatestep where the silver salt and the developer are added together orseparately to the viewing window area 82 where the test zone 83 islocated. If there is no viewing window area 82, the silver salt and thedeveloper are preferably added to the test zone 83 of the strip. In someof these embodiments, the silver enhancement is added to the test stripwhile it is still wet or dried after the use. In some of theseembodiments, the strip is removed from any housing and a portion of thestrip containing the test zone 83 is cut and treated with silverenhancement together or separately.

In one preferred embodiment, after the test is run, the strip is allowedto dry in air. Moderate drying of the strip is accomplished inapproximately 20 to 30 minutes, but is dependent upon environmentalconditions. After the strip has dried, a drop or two of the silver saltand the developers are added to the viewing window area 82 where thetest zone 83 is located. If there is no viewing window area 82, thesilver salt and the developer are preferably added to the test zone 83of the strip. This enhances the sensitivity at least 5 fold. The silversalt and the developers may be added together or separately. The silverenhancement occurs almost instantaneously and the results are preferablyread within two to three minutes after the additional of the silverenhancement. If the results are not read quickly, the strip may turnblack and the background will interfere with the reading of theresulting grey/black test line. This background can be largely minimizedif a washing solution is added to the viewing window 82/test zone 83.Sensitivity may be further enhanced with the use of a portable opticalreader, for example a miniature spectrometer made by Ocean Optics, Inc.(Dunedin, Fla.). A portable reader is a hand-held miniaturespectrometer, which quantifies the color intensity of the test linemeasuring the absorbance or the reflectance of the labeled complex whichbinds to the test line. The quantification of the test line can bedetermined by the use of a standard curve. In developing a standardcurve, one creates several titrations of the analyte concentration andrecords the reader output at each titration. The reader increases thesensitivity of a test by 5 to 10 fold. In operation, the detectionwindow to view the visible test line is placed directly on or inproximity to the spectrometer aperture so that a direct absorbance orreflectance measurement can be made.

In another preferred embodiment, the silver salt and/or developersolution includes a volatile liquid. The silver salt and developer couldbe made up together in a single solution or as separate solutions. Anyliquid that evaporates at room temperature or vaporizes easily and doesnot interfere with the test could be used. The volatile solvent ischosen in such a way that it does not dissolve the membrane material(e.g. nitrocellulose) that makes up the test zone 83 where the secondbinding partner 17 (see FIG. 1), 38 (see FIGS. 3A-3C and 7A) or theimmobilized tag 50 (see FIGS. 4A-4C, 5A-5B, 6A-6B and 7B-7D) arelocated. Some examples of a volatile liquid that could be used include,but are not limited to, methanol, isopropyl alcohol, low concentrationsof benzene, and low concentrations of acetone. The silver enhancementhas the silver salt and a developer which is preferably relativelyorganic in nature. The silver salt and developer solution are added tothe viewing window area 82 where the test zone 83 is located at the endof the test (for example approximately 10 minutes after the sample wasadded to the strip), when the strip is still quite wet. If there is noviewing window area 82, the silver salt and the developer are preferablyadded to the test zone 83 of the strip. The volatile liquid “dries” thearea where the liquid is added (the test zone 83). In this embodiment,it is not necessary to wait for the entire strip to be moderately dry.This embodiment creates “in-situ” drying of only the area of interest(the test zone 83).

In some embodiments as shown in FIG. 12, the amplification is due to a“stacking” phenomenon where a second conjugate 74 “stacks” on at least aportion of the complex formed during the assay. In these embodiments,the first conjugate 72 includes an additional portion 73 to which abinding partner 76 of the portion 73 specifically binds, and the secondconjugate 74 preferably also includes a label 78. For example, when thesecond binding partner 38 includes an avidin tag 39, the full sandwichis captured in the test zone by immobilized biotin 50, and subsequentlyor concurrently, the “stacking” conjugate accumulates or gets stackedonto the immobilized full sandwich at the test zone, giving rise to morestacked accumulation and better signal perception. In one embodiment,the first conjugate is gold conjugated to an antibody of the analyte andchicken IgY, and the second conjugate is a red latex bead conjugated toa rabbit anti-chicken antigen.

Preferably, “stacking” is only used in embodiments where the “fullsandwich” is formed prior to reaching the test zone. For example, inFIGS. 4C, 5B, 6B, 7B, 7C, and 7D, a full sandwich is formed in thesample application zone. In a preferred embodiment, mouse antibody onlabeled conjugate binds to the antigen to form a first complex. Thefirst complex immediately binds to the mobilized biotin labeledpolyclonal antibody to form a full sandwich as a second complex. Thesecond complex is then captured at the test zone by avidin via thebiotin label. Slower released anti-mouse label conjugate then binds andstacks on to the mouse antibody in the second complex in the test zone.The anti-mouse label conjugate is preferably located such that itreaches the test zone after the analyte complexes have formed. Somepreferred locations for the anti-mouse label conjugate include in thesample application zone, upstream of the sample application zone, addedto the buffer after a predetermined amount of time, applied to the testzone after the sandwich has been formed, or in the flow path butencapsulated to delay its release, for example, by 20 to 30 seconds. Inthis embodiment, the stacking increases the sensitivity of the assay 3-5fold.

In embodiments of the present invention with gold conjugates, which maybe used in all lateral flow assays, labeled and dried anti-chicken IgY,or another nonspecific immunogenic moiety, is incorporated on the teststrip upstream from the sample application zone or alternatively in thebuffer. When the sample is mammalian (e.g., human), the nonspecificimmunogenic moiety is preferably from a non-mammalian organism such as,for example, a bird, a fish, or a plant, so that it does not interferewith analyte binding. The second conjugate, e.g. anti-chicken IgY, isthen mobilized by the buffer. Delaying the mobilization of the secondconjugate allows the full sandwich to flow and begin binding viatag-immobilized tag, e.g. biotin-avidin, capture at the test zone in thecase of a mobile second binding partner. The full sandwich accumulatesat the test zone followed by binding and stacking of the secondconjugate, e.g. red latex beads, on top of the first conjugate, e.g.gold. This embodiment also increases the sensitivity of the assay 3-5fold. In embodiments where the second binding partner for the analyte isimmobilized at the test zone, the half sandwich preferably travels tothe test zone followed by binding and stacking.

FIG. 11 shows non-specific amplification and FIG. 12 shows specificamplification. In other embodiments, combinations of both specificamplification and non-specific amplification could be used, to furtheramplify the signal. As an example, the first amplification is due to a“stacking” phenomenon as shown and discussed above with respect to FIG.12 where a second conjugate 74 “stacks” on at least a portion of thecomplex formed during the assay. Further amplification is provided whenan amplification source 70 non-specifically deposits itself onto theconjugate such that multiple conjugates are associated with one analytebound in the test zone, as shown and discussed above with respect toFIG. 11. Other combinations of specific and non-specific amplificationcould alternatively be used.

In another embodiment of stacking and signal enhancement, enhancement isperformed using an enzyme conjugated to the stacking moiety. In oneexample, the enzyme is horseradish peroxidase, and it is conjugated to arabbit anti-mouse antibody. While horseradish peroxidase is often usedto amplify a weak signal, other enzymes that enhance weak signals couldalternatively be used including, but not limited to, alkalinephosphatase, catalase, urease, and glucose oxidase. Similarly, otherantibodies that bind to the conjugate or an intermediary couldalternatively be used. There are no nanoparticles or microspheres inthis embodiment. Instead, this embodiment includes a “soluble” form ofthe conjugate. The location where this enzyme conjugate is dried canvary; it can be upstream, downstream, or overlapping the sampleapplication zone. In embodiments with a sample compressor, the enzymeconjugate could alternatively be on the sample compressor. The enzymeconjugate is preferably dried on the test strip, but not immobilized. Itcan be located alone or in combination with other components that formthe “sandwich” with the antibody (which is preferably biotinylated)and/or the gold-conjugated antibody.

FIG. 14 shows an embodiment of a detector with an enzyme conjugated tothe stacking moiety. The control zone 46 includes an immobilized firstcontrol binding partner 110. The test zone 45 includes an immobilizedfirst test zone binding partner 109 on the membrane. A first analytebinding partner 102 conjugated to a second test zone binding partner 101is dried or otherwise incorporated (e.g., lyophilized) into the sampleapplication zone 44. While not shown in this figure, the first analytebinding partner 102 could alternatively be located upstream ordownstream of the sample application zone 44. A binding partner 107 fora second analyte binding partner 103 is conjugated to an enzyme 108, andis located upstream of the sample application zone 44. Alternatively,the binding partner 107 for the second analyte binding partner 103 couldoverlap the sample application zone 44 or be located downstream of thesample application zone 44. The pad 33 on the sample compressor 30 ispreferably embedded with the second analyte binding partner 103conjugated to a first detectable label 104 and is preferably mixed witha second control binding partner 105 conjugated to a second detectablelabel 106, which serves as a control.

While FIG. 14 shows the different reagents in certain locations on thetest strip or the sample compressor 30, other locations for each of thefirst analyte binding partner 102 conjugated to a second test zonebinding partner 101, the binding partner 107 for the second analytebinding partner 103, the second analyte binding partner conjugated tothe first detectable label 104, and the second control binding partner105 conjugated to the second detectable label 106 on the test stripand/or on the pad 33 of the sample compressor 30 are also possible.Other embodiments do not require a sample compressor 30. In theseembodiments, the reagents 101, 102, 103, 104, 105, 106, 107, and 108will be located in various locations, preferably upstream of the testzone 45, on the test strip.

The sample is taken on a sample swab 35, which is then placed on thesample application zone 44 through the sample window 81 (in embodimentswith a housing and a sample window) or just on the sample applicationzone 44. The sample compressor 30 is then compressed onto the sampleapplication zone 44. The absorbent tip of the sample compressor 30 ispreferably immersed in running buffer for approximately 15-30 secondsbefore removing the sample compressor 30. FIGS. 15A and 15B show thedifferent complexes that form between the test reagents and the analyte.If the analyte 40 is present in the sample, it complexes with the firstanalyte binding partner 102 and the second analyte binding partner 103,which complexes with the binding partner 107 conjugated with the enzyme108.

If the analyte 40 is not present in the sample, the second analytebinding partner 103 still complexes with the binding partner 107conjugated with the enzyme 108, but they do not complex with the sampleor the first analyte binding partner 102. The second test zone bindingpartner 101 will bind to the first test zone binding partner 109 in thetest zone 45, regardless of whether or not the analyte 40 is present inthe sample. However, if there is no analyte present, nothing will bevisible at the test line. The result is visually read at approximatelyten minutes. If a visible test line forms along with a visible controlline, the result indicates high levels of analyte in the sample. If, atthe end of 10 minutes, there is no visible line at the test line, thenone drop of a substrate for the enzyme is added at the test line. Ifaddition of the enzyme substrate results in a visible signal, the resultindicates a weak positive sample. A visible line at the control lineindicates that the second control binding partner 105 conjugated to thesecond detectable label 106 has bound to the first control bindingpartner 110 in the control zone 46 and that the test has run correctly.FIG. 15C shows the complex that forms in the control zone.

As an example, a Herpes Simplex Virus (HSV) detector includes thefollowing sections, as shown in FIG. 14. The control zone 46 includesimmobilized rabbit anti chicken IgY antibody 110. The test zone 45includes immobilized NeutrAvidin 109 on the nitrocellulose membrane.Biotinylated 101 polyclonal anti HSV-1 and/or HSV-2 102 is dried ontothe sample application zone 44. While not shown in this figure, the antiHSV-1/HSV-2 102 could alternatively be dried upstream or downstream ofthe sample application zone 44. Rabbit anti-mouse IgG (H&L) 107conjugated to horseradish peroxidase (HRP) 108 is dried upstream of thesample application zone 44. Alternatively, the rabbit-anti-mouse IgG 107conjugated to horseradish peroxidase 108 could overlap the sampleapplication zone 44 or be located downstream of the sample applicationzone 44. The pad 33 on the sample compressor 30 is preferably embeddedwith mouse monoclonal anti gD 1&2 103 (monoclonal antibodies directedagainst glycoprotein D of herpes simplex virus) conjugated to colloidalgold 104 and mixed with chicken IgY 105 conjugated to blue dyed latexbeads 106, which serves as a control.

The sample is taken on a sample swab 35, which is then placed on thesample application zone 44 through the sample window 81 (in embodimentswith a housing and a sample window) or just on the sample applicationzone 44. The sample compressor 30 is then compressed onto the sampleapplication zone 44. The absorbent tip of the sample compressor 30 ispreferably immersed in running buffer for approximately 15-30 secondsbefore removing the sample compressor 30. FIGS. 15A and 15B show thedifferent complexes that form between the test reagents and the analyte.If HSV (the analyte 40) is present in the sample, it complexes with thebiotinylated 101 polyclonal anti HSV1/2 102 and the mouse monoclonalanti gD 1&2 103 conjugated to colloidal gold 104, which complexes withthe rabbit anti-mouse IgG 107 conjugated with HRP 108.

If HSV is not present in the sample, the mouse monoclonal anti gD 1&2103 conjugated to colloidal gold 104 still complexes with the rabbitanti-mouse IgG 107 conjugated with HRP 108, but they do not complex withthe sample or the biotinylated 101 polyclonal anti HSV1/2 102. Thebiotinylated 101 polyclonal anti HSV1/2 102 will bind to neutravidin 109in the test zone 45, regardless of whether or not HSV is present in thesample. However, if there is no HSV present, the biotinylated 101polyclonal anti HSV1/2 102 will not be visible at the test line. Theresult is visually read at approximately ten minutes. If a visible redtest line forms along with the blue control line, the result indicateshigh levels of HSV in the sample. If, at the end of 10 minutes, there isno visible red line at the test line, then one drop of the enzymesubstrate TMBM (or another substrate for horseradish peroxidase) isadded at the test line. If addition of the TMBM results in a blue/purpletest line, the result indicates a weak positive sample. A blue line atthe control line indicates that the chicken IgY 105 conjugated to theblue dyed latex beads 106 has bound to the rabbit anti-chicken IgY 110in the control zone 46 and that the test has run correctly. FIG. 15Cshows the complex that forms in the control zone.

In this embodiment, the point of care test becomes enzyme-linked and theamplification depends on the amount of enzyme and substrate, andincreases with time. This does not happen in visually tagged conjugatesto nanoparticles like colloidal gold or microspheres like latex beads.In addition, the test line result is not due to any antigen-antibodyimmunoassay, but a binding assay between a ligand and a receptor such asneutravidin and biotin. The binding at the test line is not due toimmunological binding but chemical binding. Thus it is not anenzyme-linked immunoassay (ELISA or EIA). Instead, it is anenzyme-linked chromofiltography, or direct multiplanar enzymechromofiltography when used with a sample compressor. Even with anadditional step of adding the enzyme substrate to the test line, thetest is still simple to perform.

In an alternative stacking embodiment, shown in FIGS. 16, 17A, and 17B,an enzyme is physically bound to the detectable label on both theconjugate and the stacking moiety. In one example, the enzyme coatsvisibly detectable beads (for example, red latex beads) and isconjugated to a rabbit anti-mouse antibody. While horseradish peroxidaseis often used to amplify a weak signal, other enzymes that enhance weaksignals could alternatively be used including, but not limited to,alkaline phosphatase, catalase, urease, and glucose oxidase. Similarly,other antibodies that bind to the conjugate or an intermediary couldalternatively be used. There are no nanoparticles or microspheres inthis embodiment. Instead, this embodiment includes a “soluble” form ofthe conjugate. The location where this enzyme conjugate is dried canvary; it can be upstream, downstream, or overlapping the sampleapplication zone. In embodiments with a sample compressor, the enzymeconjugate could alternatively be on the sample compressor. The enzymeconjugate is preferably dried on the test strip, but not immobilized. Itcan be located alone or in combination with other components that formthe “sandwich” with the antibody, which is preferably biotinylated.

FIG. 16 shows an embodiment of a detector with an enzyme physicallybound to the detectable label on both the conjugate and the stackingmoiety. The control zone 46 includes an immobilized first controlbinding partner 110, similar to the detector shown in FIG. 14. The testzone 45 includes an immobilized first test zone binding partner 209 on amembrane. A first analyte binding partner 202 conjugated to a secondtest zone binding partner 201 is dried or otherwise incorporated (e.g.,lyophilized) into the sample application zone 44. While not shown inthis figure, the first analyte binding partner 202 could alternativelybe located upstream or downstream of the sample application zone 44.

A binding partner 207 for a second analyte binding partner 203, which isconjugated to an enzyme 208 and conjugated to a detectable label 215(which is also conjugated to the enzyme 208), is preferably embeddedinto the pad 33 the sample compressor 30. In other embodiments, there isonly a binding partner 207 for the second analyte binding partner 203conjugated to a detectable label 215, and the detectable label is alsoconjugated to the enzyme 208. In some embodiments, the enzyme 208 isconjugated to the detectable label 215 by coating the detectable label215. In some embodiments, the binding partner 207 conjugated to theenzyme 208 plus the binding partner 207 conjugated to the detectablelabel 215 (which is also conjugated to the enzyme 208) could be locatedon the test strip, overlapping the sample application zone 44 or beinglocated downstream or upstream of the sample application zone 44. Thepad 33 on the sample compressor 30 is preferably also embedded with thesecond analyte binding partner 203 conjugated to a detectable label 204coated with the enzyme 208, which is preferably mixed with a secondcontrol binding partner 105 conjugated to a detectable label 106 (shownin FIG. 14), which serves as a control.

While FIG. 16 shows the different reagents in certain locations on thetest strip or the sample compressor 30, other locations for each of thefirst analyte binding partner 202 conjugated to the second test zonebinding partner 201, the binding partner 207 conjugated to the enzyme208 plus the binding partner 207 conjugated to the detectable label 215coated with the enzyme, and the second control binding partner 105conjugated to the detectable label 106, on the test strip and/or on thepad 33 of the sample compressor 30 are also possible. Other embodimentsdo not require a sample compressor 30. In these embodiments, thereagents 201, 202, 203, 204, 105, 106, 207, 208, and 215 will be locatedin various locations, preferably upstream of the test zone 45, on thetest strip.

The sample is taken on a sample swab 35, which is then placed on thesample application zone 44 through the sample window 81 (in embodimentswith a housing and a sample window) or just on the sample applicationzone 44. The sample compressor 30 is then compressed onto the sampleapplication zone 44. The absorbent tip of the sample compressor 30 ispreferably immersed in running buffer for approximately 15-30 secondsbefore removing the sample compressor 30. FIGS. 17A and 17B show thedifferent complexes that form between the test reagents and the analyte.If the analyte 40 is present in the sample, it complexes with the firstanalyte binding partner 202 and the second analyte binding partner 203.The second analyte binding partner also complexes with the bindingpartner 207.

If the analyte 40 is not present in the sample, the second analytebinding partner 203 still complexes with the binding partner 207, butthey do not complex with the sample or the first analyte binding partner202. The second test zone binding partner 201 will bind to the firsttest zone binding partner 209 in the test zone 45, regardless of whetheror not the analyte 40 is present in the sample. However, if there is noanalyte 40 present, the second test zone binding partner 201 conjugatedto the first analyte binding partner 202 and complexed with the firsttest zone binding partner 209 will not be visible at the test line. Theresult is visually read at approximately ten minutes. If a visible testline forms along with a visible control line, the result indicates highlevels of analyte in the sample. If, at the end of 10 minutes, there isno visible line at the test line, then one drop of the enzyme substrateis added at the test line. If addition of the enzyme substrate resultsin a visible test line, the result indicates a weak positive sample. Avisible line at the control line indicates that the second controlbinding partner 105 has bound to the first control binding partner 110in the control zone 46 and that the test has run correctly. The controlline complex is shown in FIG. 15C.

In this embodiment, the enzyme is physically bound to the detectablelabel (for example, latex beads) and moves with the detectable label.Thus, specificity and background issues are improved. At high levels ofantigen, a positive result is easily visibly detectable by a visibleline. At very low levels, the enzyme substrate is added to the resultswindow to get an enzyme-amplified color reaction. By depositing many ofthe reagents, including the binding partner 207, which includes theenzyme 208 and the detectable label 215, on the sample compressor, thesereagents are not on the strip. In some preferred embodiments, the secondanalyte binding partner 203 can be premixed with the binding partner 207(with or without the enzyme labeled binding partner) and be embedded inthe sample compressor pad. In these embodiments, the test strip includesthe second test zone binding partner 202, which binds to the first testzone binding partner 209. This makes the test strip into a binding assayand not an immunoassay.

As an example, a Herpes Simplex Virus (HSV) detector includes thefollowing sections, as shown in FIG. 16. The control zone 46 includesimmobilized rabbit anti chicken IgY antibody 110, similar to thedetector shown in FIG. 14. The test zone 45 includes immobilizedNeutrAvidin 209 on the nitrocellulose membrane. Biotinylated 201polyclonal anti HSV-1 and/or HSV-2 202 is dried onto the sampleapplication zone 44. While not shown in this figure, the antiHSV-1/HSV-2 202 could alternatively be dried upstream or downstream ofthe sample application zone 44. Rabbit anti-mouse IgG (H&L) 207conjugated to horseradish peroxidase (HRP) 208 plus rabbit anti-mouseIgG 207 conjugated to red latex beads 215 coated with horseradishperoxidase and is preferably embedded into the pad 33 the samplecompressor 30. In other embodiments, there is only rabbit anti-mouse IgG207 conjugated to red latex beads 215 coated with horseradishperoxidase. Alternatively, the rabbit-anti-mouse IgG 207 conjugated tohorseradish peroxidase 208 plus rabbit anti-mouse IgG 207 conjugated tored latex beads 215 coated with horseradish peroxidase could be locatedon the test strip, overlapping the sample application zone 44 or beinglocated downstream or upstream of the sample application zone 44. Thepad 33 on the sample compressor 30 is preferably also embedded withmouse monoclonal anti gD 1&2 203 (monoclonal antibodies directed againstglycoprotein D of herpes simplex virus) conjugated to red latex beads204 coated with horseradish peroxidase and mixed with chicken IgY 105conjugated to blue dyed latex beads 106 (shown in FIG. 14), which servesas a control.

The sample is taken on a sample swab 35, which is then placed on thesample application zone 44 through the sample window 81 (in embodimentswith a housing and a sample window) or just on the sample applicationzone 44. The sample compressor 30 is then compressed onto the sampleapplication zone 44. The absorbent tip of the sample compressor 30 ispreferably immersed in running buffer for approximately 15-30 secondsbefore removing the sample compressor 30. FIGS. 17A and 17B show thedifferent complexes that form between the test reagents and the analyte.If HSV (the analyte 40) is present in the sample, it complexes with thebiotinylated 201 polyclonal anti HSV1/2 202 and the mouse monoclonalanti gD 1&2 203 conjugated to red latex beads 204, which complexes withthe rabbit anti-mouse IgG 207 conjugated with HRP 208 and the rabbitanti-mouse IgG 207 conjugated to red latex beads 215 coated withhorseradish peroxidase.

If HSV is not present in the sample, the mouse monoclonal anti gD 1&2203 conjugated to the red latex beads 204 still complexes with therabbit anti-mouse IgG 207, but they do not complex with the sample orthe biotinylated 201 polyclonal anti HSV1/2 202. The biotinylated 201polyclonal anti HSV1/2 202 will bind to NeutrAvidin 209 in the test zone45, regardless of whether or not HSV is present in the sample. However,if there is no HSV present, the biotinylated 201 polyclonal anti HSV 1/2202 will not be visual at the test line. The result is visually read atapproximately ten minutes. If a visible red test line forms along withthe blue control line, the result indicates high levels of HSV in thesample. If, at the end of 10 minutes, there is no visible red line atthe test line, then one drop of the enzyme substrate TMBM (or anothersubstrate for horseradish peroxidase) is added at the test line. Ifaddition of the TMBM results in a blue/purple test line, the resultindicates a weak positive sample. A blue line at the control lineindicates that the chicken IgY 105 conjugated to the blue dyed latexbeads 106 has bound to the rabbit anti-chicken IgY 110 in the controlzone 46 and that the test has run correctly. The control line complex isshown in FIG. 15C.

In this example, rabbit anti mouse antibody is conjugated to the enzyme,which is also conjugated to the red latex beads, and additional rabbitanti-mouse antibody is conjugated directly to the same beads. The enzymeis physically bound to the beads and moves with the beads. Thus,specificity and background issues are improved. At high levels ofantigen, a positive result is easily visibly detectable by a red line.At very low levels, the enzyme substrate is added to the results windowto get an enzyme-amplified color reaction.

By depositing the rabbit anti mouse antibody conjugated to the red beads(along with the enzyme conjugate on the same bead) on the samplecompressor, these reagents are not on the strip. In some preferredembodiments, the free mouse monoclonal anti gD 1&2 can be premixed withthe rabbit anti mouse (with or without the enzyme labeled rabbit antimouse) and be embedded in the sample compressor pad. In theseembodiments, the test strip includes biotin which binds to neutravidin.This makes the test strip into a binding assay and not an immunoassay.

FIGS. 18, 19A, and 19B show another stacking embodiment of the presentinvention. In this embodiment, an enzyme is conjugated/physically boundto a detectable label on the stacking moiety and the conjugate thatbinds to the analyte does not include a detectable label. Thisembodiment further increases specificity. In one example, the enzymecoats visibly detectable beads (for example, red latex beads) and isconjugated to a rabbit anti-mouse antibody. While horseradish peroxidaseis often used to amplify a weak signal, other enzymes that enhance weaksignals could alternatively be used including, but not limited to,alkaline phosphatase, catalase, urease, and glucose oxidase. Similarly,other antibodies that bind to the conjugate or an intermediary couldalternatively be used. There are no nanoparticles or microspheres inthis embodiment. Instead, this embodiment includes a “soluble” form ofthe conjugate. The location where this enzyme conjugate is dried canvary; it can be upstream, downstream, or overlapping the sampleapplication zone. In embodiments with a sample compressor, the enzymeconjugate could alternatively be on the sample compressor. The enzymeconjugate is preferably dried on the test strip, but not immobilized. Itcan be located alone or in combination with other components that formthe “sandwich” with the antibody (which is preferably biotinylated).

An embodiment of a detector with enzyme conjugated/physically bound to adetectable label on the stacking moiety and a conjugate that binds tothe analyte that does not include a detectable label is shown in FIG.18. The control zone 46 includes an immobilized first control bindingpartner 110, similar to the detector shown in FIG. 14. The test zone 45includes an immobilized first test zone binding partner 309 on amembrane. A first analyte binding partner 302 conjugated to a secondtest zone binding partner 301 is dried or otherwise incorporated (e.g.,lyophilized) into the sample application zone 44. While not shown inthis figure, the first analyte binding partner 302 could alternativelybe located upstream or downstream of the sample application zone 44. Amixture of a binding partner 307 for the second analyte binding partner303 conjugated to an enzyme 308 and the binding partner 307 conjugatedto a detectable label 315 (for example, latex beads) coated or otherwiseconjugated to the enzyme 308 is preferably embedded into the pad 33 ofthe sample compressor 30. In other embodiments, there is only thebinding partner 307 conjugated to the detectable label 315, which isalso conjugated to the enzyme 308 (for example, by the enzyme coatinglatex beads). While the binding partner 307 conjugated to the enzyme andthe binding partner 307 conjugated to the detectable label 315 coatedwith the enzyme is shown on the sample compressor 30 in this figure,these components could alternatively be located on the test strip,overlapping the sample application zone 44 or being located downstreamor upstream of the sample application zone 44. The pad 33 on the samplecompressor 30 is preferably also embedded with a second analyte bindingpartner 303. Unlike in the previous embodiments, the second analytebinding partner 303 is not conjugated to a detectable label or anenzyme. In some embodiments, the second analyte binding partner 303 ispreferably mixed with the second control binding partner 105 conjugatedto the detectable label 106 (shown in FIG. 14), which serves as acontrol.

While FIG. 18 shows the different reagents in certain locations on thetest strip or the sample compressor 30, other locations for each of thefirst analyte binding partner 302 conjugated to the second test zonebinding partner 301, the mixture of the binding partner 307 for thesecond analyte binding partner 303 conjugated to an enzyme 308 and thebinding partner 307 conjugated to a detectable label 315 coated orotherwise conjugated to the enzyme 308, the second analyte bindingpartner 303 and the second control binding partner 105 conjugated to adetectable label 106, on the test strip and/or on the pad 33 of thesample compressor 30 are also possible. Other embodiments do not requirea sample compressor 30. In these embodiments, the reagents 301, 302,303, 304, 105, 106, 307, 308, and 315 will be located in variouslocations, preferably upstream of the test zone 45, on the test strip.

The sample is taken on a sample swab 35, which is then placed on thesample application zone 44 through the sample window 81 (in embodimentswith a housing and a sample window) or just on the sample applicationzone 44. The sample compressor 30 is then compressed onto the sampleapplication zone 44. The absorbent tip of the sample compressor 30 ispreferably immersed in running buffer for approximately 15-30 secondsbefore removing the sample compressor 30. FIGS. 19A and 19B show thedifferent complexes that form between the test reagents and the analyte.If the analyte 40 is present in the sample, it complexes with the firstanalyte binding partner 302 and the second analyte binding partner 303.The second analyte binding partner 303 also complexes with the bindingpartner 307.

If the analyte 40 is not present in the sample, the second analytebinding partner 303 still complexes with the binding partner 307, butthey do not complex with the sample or the first analyte binding partner302. The second test zone binding partner 301 binds to the first testzone binding partner 309 in the test zone 45, regardless of whether ornot the analyte is present in the sample. However, if there is noanalyte 40 present, the resulting complex will not be visible at thetest line. The result is visually read at approximately ten minutes. Ifa visible test line forms along with the visible control line, theresult indicates high levels of analyte 40 in the sample. If, at the endof 10 minutes, there is no visible line at the test line, then one dropof an enzyme substrate is added at the test line. If addition of theenzyme substrate results in a visible test line, the result indicates aweak positive sample. A visible line at the control line indicates thatthe second control binding partner 105 conjugated to the detectablelabel 106 has bound to the first control binding partner 110 in thecontrol zone 46 and that the test has run correctly. The control linecomplex is shown in FIG. 15C.

In this embodiment, the binding partner 307 is conjugated to the enzyme308, which is also conjugated to the detectable label 315 (for example,latex beads), and additional binding partner 307 is conjugated directlyto the same detectable label 315. The enzyme is physically bound to thedetectable label and moves with the detectable label. Thus, specificityand background issues are improved. At high levels of antigen, apositive result is easily visibly detectable by a visible line. At verylow levels, the enzyme substrate is added to the results window to getan enzyme-amplified color reaction.

By depositing the binding partner 307 and its other components (308 and315) on the sample compressor, these reagents are not on the strip. Insome preferred embodiments, the second analyte binding partner 303 canbe premixed with the binding partner 307 (with or without the enzymelabeled binding partner 307) and be embedded in the sample compressorpad. In these embodiments, the device includes binding partners such asbiotin and avidin. This makes the test strip into a binding assay andnot an immunoassay.

As an example, a Herpes Simplex Virus (HSV) detector includes thefollowing sections, as shown in FIG. 18. The control zone 46 includesimmobilized rabbit anti-chicken IgY antibody 110, similar to thedetector shown in FIG. 14. The test zone 45 includes immobilizedneutravidin 309 on a nitrocellulose membrane. Biotinylated 301polyclonal anti HSV-1 and/or HSV-2 302 is dried onto the sampleapplication zone 44. While not shown in this figure, the antiHSV-1/HSV-2 302 could alternatively be located upstream or downstream ofthe sample application zone. Rabbit anti-mouse IgG (H&L) 307 conjugatedto horseradish peroxidase (HRP) 308 plus rabbit anti-mouse IgG 307conjugated to red latex beads 315 coated with horseradish peroxidase ispreferably embedded into the pad 33 of the sample compressor. In otherembodiments, there is only rabbit anti-mouse IgG 307 conjugated to redlatex beads 315 coated with horseradish peroxidase. Alternatively, therabbit anti-mouse IgG 307 conjugated to horseradish peroxidase 308 plusrabbit anti-mouse IgG 307 conjugated to red latex beads coated withhorseradish peroxidase 308 could be located on the test strip,overlapping the sample application zone 44 or being located downstreamor upstream of the sample application zone 44. The pad on the samplecompressor 30 is preferably also embedded with free mouse monoclonalanti gD 1&2 303. Unlike in the previous embodiments, the free mousemonoclonal antibodies 303 are not conjugated to a detectable label or anenzyme. The free mouse monoclonal antibodies 303 are preferably mixedwith chicken IgY 105 conjugated to blue dyed latex beads (shown in FIG.14), which serves as a control.

The sample is taken on a sample swab 35, which is then placed on thesample application zone 44 through the sample window 81 (in embodimentswith a housing and a sample window) or just on the sample applicationzone 44. The sample compressor 30 is then compressed onto the sampleapplication zone 44. The absorbent tip of the sample compressor 30 ispreferably immersed in running buffer for approximately 15-30 secondsbefore removing the sample compressor 30. FIGS. 19A and 19B show thedifferent complexes that form between the test reagents and the analyte.If HSV (the analyte 40) is present in the sample, it complexes with thebiotinylated 301 polyclonal anti HSV1/2 302 and the mouse monoclonalanti gD 1&2 303, which complexes with the rabbit anti-mouse IgG 307conjugated with HRP 308 and the rabbit anti-mouse IgG 307 conjugated tored latex beads 315 coated with horseradish peroxidase.

If HSV is not present in the sample, the mouse monoclonal anti gD1&2 303still complexes with the rabbit anti-mouse IgG 307, but they do notcomplex with the sample or the biotinylated 301 polyclonal anti HSV1/2302. The biotinylated 301 polyclonal anti HSV1/2 202 will bind toneutravidin 309 in the test zone 45, regardless of whether or not HSV ispresent in the sample. However, if there is no HSV present, thebiotinylated 301 polyclonal anti HSV 1/2 302 will not be visible at thetest line. The result is visually read at approximately ten minutes. Ifa visible red test line forms along with the blue control line, theresult indicates high levels of HSV in the sample. If, at the end of 10minutes, there is no visible red line at the test line, then one drop ofthe enzyme substrate TMBM (or another substrate for horseradishperoxidase) is added at the test line. If addition of the TMBM resultsin a blue/purple test line, the result indicates a weak positive sample.A blue line at the control line indicates that the chicken IgY 105conjugated to the blue dyed latex beads 106 has bound to the rabbitanti-chicken IgY 110 in the control zone 46 and that the test has runcorrectly. The control line complex is shown in FIG. 15C.

In this example, rabbit anti mouse antibody is conjugated to the enzyme,which is also conjugated to the red latex beads, and additional rabbitanti-mouse antibody is conjugated directly to the same beads. The enzymeis physically bound to the beads and moves with the beads. Thus,specificity and background issues are improved. At high levels ofantigen, a positive result is easily visibly detectable by a red line.At very low levels, the enzyme substrate is added to the results windowto get an enzyme-amplified color reaction.

By depositing the rabbit anti mouse antibody conjugated to the red beads(along with the enzyme conjugate on the same bead) on the samplecompressor, these reagents are not on the strip. In some preferredembodiments, the free mouse monoclonal anti gD 1&2 can be premixed withthe Rabbit anti mouse (with or without the enzyme labeled Rabbit antimouse) and be embedded in the sample compressor pad. In theseembodiments, the device includes binding partners such as biotin andavidin. This makes the test strip into a binding assay and not animmunoassay.

In some preferred embodiments, the nitrocellulose is “blocked” withblockers, which increases the specificity of the reaction. Some examplesfor blockers include, but are not limited to, casein, and Bovine SerumAlbumin (BSA). Whenever one blocks the nitrocellulose membrane, theinherent charge of the nitrocellulose is neutralized and thus, noadditional protein can bind to the blocked membrane. In addition, thechromatographic structure is changed and the flow is more like a glidingor sliding flow instead of traditional chromatography. The result is aunique chromofiltography process.

FIG. 21A shows another embodiment of a lateral flow test strip withenhancing elements. This embodiment preferably includes a labeledbinding partner 407 that is specific for a species instead of an analyte40. As an example, when the binding partner 402 for the analyte is amouse antibody, the labeled species specific binding partner 407 is ananti-mouse antibody. As another example, when the binding partner 402for the analyte is a rabbit antibody, the labeled species specificbinding partner 407 is an anti-rabbit antibody. Those skilled in the artwould understand that any species specific binding partner 407, or otherbinding partner not specific for the analyte 40 but specific for abinding partner 402 for the analyte, could be used in this embodiment.Those skilled in the art would also know how to choose species tominimize cross-reactions.

The sample application zone 44 includes a first binding partner 402 forthe analyte 40. Note that the first binding partner 402 does not includea detectable label. In this embodiment, some of the first bindingpartner 402 is preferably tagged 401 and a binding partner 409 for thetag 401 is preferably labeled with a detectable label. In preferredembodiments, the amount of the first binding partner 402 that is tagged401 is from 1-10% of the total amount of the first binding partner 402in the test.

The sample application zone 44 also includes a labeled species specificbinding partner 407 (conjugated to a detectable label 417) that binds tothe first binding partner 402 due to the species of the first bindingpartner 402. The sample application zone 44 also preferably includes alabeled 415 control binding partner 405 While the first binding partner402 for the analyte 40, the conjugate including a visible label 417 anda species specific binding partner 407, and the control conjugate 405conjugated to a visible label 415 are shown in the sample applicationzone 44 in this figure, any combination of these elements may be locatedin other locations on the test strip (upstream, downstream, oroverlapping the sample application zone) or on a sample compressor 30,as described in earlier embodiments.

The test zone 45 includes an immobilized second binding partner 427 tothe analyte 40. The control zone 46 includes an immobilized bindingpartner 420 for the control binding partner 405. The test zone 45 andthe control zone 46 are preferably located on a nitrocellulose membrane.

When a sample including analyte 40 is added to the test strip, the firstbinding partner 402 binds to the analyte 40 and forms a “half sandwich”.This preferably occurs without flow on the test strip. When runningbuffer is applied, it mobilizes the “half sandwich”. The running bufferalso mobilizes the species specific binding partner 407. During flow,the species specific binding partner 407 interacts with and binds to thefirst binding partner 402 in the half sandwich. Due to multiple bindingsites on the first binding partner 402, there is an aggregation orstacking effect that enhances the detection of the analyte 40. In thetest zone 45, the analyte 40, which is now part of an aggregate orstacked complex, binds to the immobilized second binding partner 427 toform the full sandwich. The result is an enhanced visible signal formedin the test zone 45. Binding between the control binding partner 405 andthe immobilized control binding partner 420 results in a detectablesignal 415.

In the presence of the analyte 40, the detectable signal 417 conjugatedto the species specific binding partner 407 is part of the complex andshould be visible. If a visible test line is “read” by the user, thetest is recorded as a positive result for the presence of the analyte40. If the test line is not visible or equivocal, then one or more dropsof a fluid including a tag binding partner 409 for the tag 401conjugated to a detectable label (for example, colloidal gold or latexbeads) is added in the test zone 45. The tag binding partner 409instantly binds to the tag 401 on the first binding partner 402. Thisgreatly enhances the visibility of the test line in the presence of theanalyte 40. In the absence of the analyte, the tag binding partner 409dissipates and no test line is visible.

FIG. 21B shows a stacked complex at the test line when analyte 40 ispresent in the sample. FIG. 21C shows the stacked complex with theaddition of the tags 401 and 409.

In an example of the embodiment shown in FIGS. 21A through 21C fordetecting Herpes Simplex Virus (HSV), the sample application zone 44includes free mouse HSV gD 1&2 402 (which binds to HSV), as well as somebiotinylated 401 mouse HSV gD1&2 402. In a preferred embodiment,approximately 1-10% of the free HSV gD1&2 402 is biotinylated 401.

The sample application zone 44 also includes rabbit anti-mouse antibody407 conjugated to red latex beads 417 and a control chicken IgY antibody405 conjugated to blue latex beads 415. Note that the rabbit anti-mouseantibody 407 is not specific to an analyte 40. Instead, it bindsspecifically to the mouse HSV gD1&2 antibody 402. As discussed above,any of the HSV gD1&2 402, the biotinylated 401 HSV gD1&2 402, the rabbitanti-mouse antibody conjugated to the red latex beads, the chicken IgYantibody conjugated to blue latex beads, or any combination of theseelements, may alternatively be upstream, downstream, or overlapping thesample application zone 44, or included on a sample compressor 30 inembodiments where a sample compressor 30 is used. The test zone 45includes immobilized rabbit anti-HSV 427, which binds to the HSV analyte40 when present in the sample. The control zone 46 includes immobilizedrabbit anti-chicken/rabbit IgG 420. The test zone 45 and the controlzone 46 are preferably located on a nitrocellulose membrane.

When a sample including analyte 40 is added to the test strip, the HSVgD1&2 402 binds to the HSV analyte 40 and forms a “half sandwich”. Thisoccurs without flow on the test strip. When running buffer is applied,it mobilizes the “half sandwich”. The running buffer also mobilizes therabbit anti-mouse antibody 407. During flow, the rabbit anti-mouseantibody 407 interacts with and binds to the HSV gD1&2 402 antibody inthe half sandwich. Due to multiple binding sites on the mouse antibody402, there is an aggregation or stacking effect that enhances thedetection of the analyte 40. In the test zone 45, the aggregate orstacked complex analyte 40, which is now part of an aggregate or stackedcomplex, binds to the immobilized rabbit anti-HSV 427 to form the fullsandwich. The result is an enhanced visible signal forming in the testzone 45. Binding occurs between the control conjugate chicken IgY 405and the immobilized rabbit anti-chicken/rabbit IgG 420, resulting in ablue detectable label 415.

In the presence of the analyte 40, the red latex beads 417 conjugated tothe rabbit anti-mouse antibody 407 are part of the complex and should bevisible. If a visible test line is “read” by the user, the test isrecorded as a positive result for the presence of the analyte 40. If thetest line is not visible or equivocal, then a drop of avidin,Neutravidin, or streptavidin conjugated 409 to colloidal gold or latexbeads is added in the test zone 45. The avidin, neutravidin, orstreptavidin conjugate 409 instantly binds to the biotin 401 on the HSVgD1&2 antibody 402. This greatly enhances the visibility of the testline in the presence of the analyte 40. In the absence of the analyte,the avidin, streptavidin, or neutravidin conjugate 409 dissipates and notest line is visible.

In some embodiments, instead of a nitrocellulose membrane, one can usemembranes such as nylon or polyester which are neutral. In theseembodiments, the proteins such as neutravidin, antibodies and antigensare not immobilized directly. They are instead, conjugated tomicrospheres which are “deposited” into the membrane and are held in thecrevices. While using a neutral membrane is shown with respect to thisparticular embodiment, neutral membranes and microspheres deposited ontothose membranes could alternatively be used in other embodiments of thepresent invention.

FIG. 13 shows some preferred locations of signal enhancement materialsfor both silver enhancement and stacking in embodiments of lateral flowdevices of the present invention. FIG. 13 schematically shows twooptions for the location of the detection zone, and only the elementsspecific to the signal enhancement are shown in the figure.

In embodiments with silver enhancement, the silver salt 70 is preferablylocated in a zone 90 between the sample application zone 44 and the testzone 45 to allow at least part of the sandwich to form before silversalt binding. Alternatively, the silver salt 70 may be placed on the pad33 of the sample compressor 30, in the sample application zone 44, in azone 92 upstream of the sample application zone 44, in the runningbuffer 43, or directly on the test zone 45 after the assay has been run.In some embodiments, the silver developer 71 is also located in the zone90 between the sample application zone and the test zone. In otherembodiments, the silver developer 71 is located in the zone 92 upstreamof the sample application zone 44, in the running buffer 43, on the pad33 of the sample compressor 30, or directly on the test zone 45 afterthe assay has been run.

In embodiments with stacking, the first conjugate 72 may be located onthe pad 33 of the sample compressor 30, in the sample application zone44, in a zone 92 upstream of the sample application zone 44, or in azone 90 downstream from the sample application zone. Alternatively, thefirst conjugate 72 may be pre-mixed with the sample prior to applicationto the sample application zone; in this embodiment, the half sandwich isformed outside of the assay device. The second conjugate 74 ispreferably located in a zone 92 upstream from the sample applicationzone. Alternatively, the second conjugate 74 may be located on the pad33 of the sample compressor 30. Alternatively, the second conjugate 74may be in a location where it can be delayed from reaching the firstconjugate 72, including, but not limited to, upstream of the sampleapplication zone, upstream of the conjugate, or added at a time afterthe assay has begun, such as in the running buffer or directly at thetest zone. Although not preferred, either or both of the first conjugate72 or the second conjugate 74 could alternatively be located in therunning buffer 43 (not shown).

Although the methods and devices are described herein as sandwichassays, methods and devices of the present invention may equally be usedin competitive assays. In these competitive assays, the conjugatepreferably includes an analyte or an analyte analog, rather than abinding partner of the analyte, bound to a label, or, alternatively, thesecond binding partner is replaced with analyte or analyte analog. Apositive test result is then indicated by the lack of the presence ofthe label in the test zone of the test strip.

Accordingly, it is to be understood that the embodiments of theinvention herein described are merely illustrative of the application ofthe principles of the invention. Reference herein to details of theillustrated embodiments is not intended to limit the scope of theclaims, which themselves recite those features regarded as essential tothe invention.

1. A lateral flow device for detecting an analyte in a samplecomprising: a sample compressor; a sample collector comprising a samplecollection portion for collection of the sample; a test strip comprisinga sample application zone and a test zone; a conjugate comprising afirst binding partner for the analyte and a label; and a second bindingpartner for the analyte; wherein a component selected from the groupconsisting of the conjugate, the second binding partner and both theconjugate and the second binding partner is not located on the teststrip prior to use of the lateral flow device; and wherein the samplecompressor, the sample collector, and the test strip form a verticalstack to apply the sample to the test strip by compression; wherein thesample collector is located between the sample compressor and the teststrip in the vertical stack.
 2. The lateral flow device of claim 1,wherein the sample compressor comprises a pad and wherein the conjugateor the second binding partner is located on the pad prior to use of thelateral flow device.
 3. The lateral flow device of claim 2 furthercomprising a first control binding partner located on the pad and asecond control binding partner immobilized in a control zone of the teststrip, wherein the first control binding partner is a binding partnerfor the second control binding partner.
 4. The lateral flow device ofclaim 2, wherein the conjugate is located on the pad and the secondbinding partner is located on an external medium.
 5. The lateral flowdevice of claim 2, wherein the second binding partner is located on thepad and the conjugate is located on an external medium.
 6. The lateralflow device of claim 1, wherein the lateral flow device is formed suchthat a positive result is only achieved by capture of the analyte in thetest zone through formation of a complex between the analyte, the firstbinding partner, and the second binding partner.
 7. The lateral flowdevice of claim 1, wherein the test zone comprises no molecule whichspecifically binds the analyte.
 8. The lateral flow device of claim 1,wherein the second binding partner comprises a tag and the test zonecomprises an immobilized binding partner of the tag.
 9. The lateral flowdevice of claim 1 further comprising a housing surrounding at least aportion of the test strip, wherein a rotatable portion of the housingforms the sample compressor.
 10. The lateral flow device of claim 1further comprising a housing surrounding at least a portion of the teststrip, wherein an insertable cartridge forms the sample compressor. 11.A universal test strip for use in a lateral flow device, the universaltest strip comprising a test zone and comprising no molecule whichspecifically binds an analyte.
 12. The universal test strip of claim 11further comprising a control zone and a control binding partnerimmobilized in the control zone.
 13. The universal test strip of claim11 further comprising a tag immobilized in the test zone.
 14. Theuniversal test strip of claim 11, wherein the tag comprises a moleculeselected from the group consisting of biotin, avidin, neutravidin,streptavidin, a lectin, and a glycosyl moiety.
 15. A sample compressorfor use in a lateral flow device, the sample compressor comprising a padand at least one binding partner on the pad, wherein the binding partnerbinds an analyte.
 16. The sample compressor of claim 15 furthercomprising a mobile control binding partner on the pad.
 17. A universalsample compressor for use in a lateral flow device, the samplecompressor comprising no molecule which specifically binds an analyte.18. The universal sample compressor of claim 17 further comprising a padand a mobile control binding partner on the pad.
 19. A lateral flowdevice for detecting an analyte comprising: a test strip comprising asample application zone and a test zone; a first conjugate comprising afirst binding partner for the analyte; a second binding partner for theanalyte; and an enhancement element; wherein the analyte, the conjugate,and the second binding partner form a sandwich which is immobilized inthe test zone when the analyte is present; and wherein the enhancementelement binds to the sandwich to increase a detection signal in the testzone.
 20. The lateral flow device of claim 19, wherein the firstconjugate further comprises a label.
 21. The device of claim 19, whereinthe first conjugate further comprises colloidal gold and the enhancementelement comprises at least one silver salt.
 22. The device of claim 19,wherein the enhancement element comprises an antigen and the firstconjugate further comprises a specific binding partner for the antigen.23. The device of claim 19, wherein the enhancement element furthercomprises a label.
 24. The lateral flow device of claim 19, wherein thetest zone comprises no molecule which specifically binds the analyte.25. The lateral flow device of claim 19, wherein the second bindingpartner comprises a tag and the test zone comprises an immobilizedbinding partner of the tag.
 26. The lateral flow device of claim 19,wherein: the first binding partner for the analyte comprises a mixtureof the first binding partner for the analyte conjugated to a tag and thefirst binding partner for the analyte without the tag; the secondbinding partner for the analyte is immobilized in the test zone; andwherein the enhancement element comprises a second conjugate comprisinga label and a binding partner for the first binding partner for theanalyte.
 27. The lateral flow device of claim 26, wherein approximately1-10% of the first binding partner for the analyte is conjugated to atag.
 28. The lateral flow device of claim 27, further comprising a tagbinding partner with a detectable label, wherein when the tag bindingpartner is added to the test at the test zone, it increases a detectionsignal in the presence of analyte.
 29. The lateral flow device of claim19, wherein the enhancement element comprises an enzyme.
 30. A method ofapplying a sample to a test strip of a lateral flow device, the methodcomprising the steps of: a) placing a sample collector comprising asample collection portion with the sample in a vertical stack between asample compressor and a sample application zone of the test strip; andb) applying a pressure to the sample collection portion using the samplecompressor to transfer at least a portion of the sample to the sampleapplication zone.
 31. The method of claim 30, wherein the samplecompressor comprises a component selected from the group consisting of afirst binding partner, a second binding partner and both the firstbinding partner and the second binding partner.
 32. The method of claim30, wherein the sample collector delivers the sample passively bycontact or through pressure onto the test strip prior to applying thesample compressor to the vertical stack.
 33. The method of claim 30,wherein step a) further comprises placing a pad with a binding partnerfor an analyte on the vertical stack and wherein in step b) at least aportion of the binding partner is transferred to the sample applicationzone.
 34. The method of claim 30, wherein step b) occurs without flow.35. A lateral flow device for detecting an analyte in a samplecomprising: a sample compressor; a sample collector comprising a samplecollection portion for collection of the sample; a test strip comprisinga sample application zone and a test zone; a conjugate comprising afirst binding partner for the analyte and a label; and a second bindingpartner for the analyte; wherein a component selected from the groupconsisting of the conjugate, the second binding partner and both theconjugate and the second binding partner is not located on the teststrip prior to use of the lateral flow device; and wherein the samplecompressor, the sample collector, and the component not on the teststrip prior to use of the lateral flow device form a vertical stack toapply the sample to the test strip by compression.
 36. The lateral flowdevice of claim 35, further comprising at least one external pad,wherein the component not on the test strip prior to use of the lateralflow device is located on the external pad.
 37. A lateral flow devicefor detecting an analyte in a sample comprising: a sample compressor; asample collector comprising a sample collection portion for collectionof the sample; a test strip comprising a sample application zone and atest zone; a conjugate comprising a first binding partner for theanalyte and a label; and a second binding partner for the analyte;wherein a component selected from the group consisting of the conjugate,the second binding partner and both the conjugate and the second bindingpartner is not located on the test strip prior to use of the lateralflow device; and wherein the conjugate and the second binding partnercome into contact with the sample after pressure transfers the sampleonto the test strip.
 38. A method of enhancing a signal on a lateralflow device comprising a test strip comprising a sample application zoneand a test zone, a conjugate comprising a first binding partner for ananalyte in a sample and a label, a second binding partner for theanalyte; and an enhancement element, comprising the steps of: a)immobilizing a sandwich formed between the analyte, if present in thesample, the conjugate, and the second binding partner in a test zone ofthe lateral flow device; and b) binding the enhancement element to thesandwich such that the enhancement element increases a detection signalin the test zone.
 39. The method of claim 38, wherein step b) comprisesthe substep of adding the enhancement element directly to the test zoneafter the sandwich has reached the test zone.
 40. The method of claim38, wherein the enhancement element is in a volatile liquid.
 41. Themethod of claim 38, wherein the conjugate further comprises colloidalgold and the enhancement element comprises at least one silver salt. 42.The method of claim 38, wherein the enhancement element comprises anantigen and the conjugate further comprises a specific binding partnerfor the antigen.
 43. The device of claim 38, wherein the enhancementelement further comprises a label.
 44. The method of claim 38, whereinthe test zone comprises no molecule which specifically binds theanalyte.
 45. A method of detecting at least one analyte in a sampleusing a point-of-care assay device, the method comprising the steps of:a) adding a sample to a sample application zone of a chromatographictest strip; b) after step a), adding at least one labeled first bindingpartner for the analyte to the sample application zone such that, if theanalyte is present in the sample, a first analyte complex forms betweenthe first binding partner and the analyte; c) exposing the sample to atleast one second tagged binding partner for the analyte; wherein, whenthe analyte is present, steps a) through c) result in the formation of asecond analyte complex between the first binding partner, the analyte,and the second binding partner; and d) capturing the second analytecomplex with an immobilized tag in a detection zone of thechromatographic test strip, wherein the immobilized tag complexes with atag portion of the second binding partner.
 46. The method of claim 45,wherein the first analyte complex forms without an addition of buffer tothe test strip.
 47. The method of claim 46, wherein the second analytecomplex forms without the addition of buffer to the test strip.
 48. Themethod of claim 45, wherein the point-of-care device comprises thechromatographic test strip, a sample compressor comprising at least thelabeled first binding partner, and a sample collector for collecting thesample, wherein the sample compressor, the sample collector, and thechromatographic test strip form a vertical stack to apply the sample tothe test strip by compression.
 49. The method of claim 48, wherein thesample collector delivers the sample passively by contact or throughpressure onto the test strip prior to applying the sample compressor tothe vertical stack.
 50. The method of claim 45, wherein a tag on thesecond binding partner and the immobilized tag comprise a pair selectedfrom the group consisting of: a) a first biotin tag and a secondimmobilized tag selected from the group consisting of avidin,neutravidin and streptavidin; b) a first tag selected from the groupconsisting of avidin, neutravidin and streptavidin and a second biotinimmobilized tag; c) a first lectin tag and a second glycosyl moietyimmobilized tag; and d) a first glyosyl moiety tag and a second lectinimmobilized tag.